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User Guide - Plexon Inc

1. omf File Name C PL2Data pl2splittest all data 64 channels_spl_001 03 omf Files File Name Start Time Duration 4 C PL2Data pl2spiittestiall data 64 channels_spl_001 pi2 Monday July 22 2013 08 33 44 00 00 10 2 C PL2Data pl2spiittest all data 64 channels_spl_002 pi2 Monday July 22 2013 08 33 54 00 00 10 3 C PL2Data pl2spliittest all data 64 channels_spl_003 pi2 Monday July 22 2013 08 34 04 00 00 10 4 C PL2Data pl2spiittestiall data 64 channels_spl_004 pi2 Monday July 22 2013 08 34 14 00 00 10 S C PL2Data pl2spiittest all data 64 channels_spl_005 pi2 Monday July 22 2013 08 34 24 00 00 10 6 C PL2Data pi2splittestiall data 64 channels_spl_006 pi2 Monday July 22 2013 08 34 34 00 00 10 7 C PL2Data pl2spiittestiall data 64 channels_spl_007 pi2 Monday July 22 2013 08 34 44 00 00 10 8 C PL2Data pl2splittest all data 64 channels_spl_008 pi2 Monday July 22 2013 08 34 54 00 00 10 9 C PL2Data pi2splittestiall data 64 channels_spl_009 pi2 Monday July 22 2013 03 35 04 00 00 10 10 C PL2Data pl2spiittest all data 64 channels_spl_010 pi2 Monday July 22 2013 08 35 14 00 00 10 Add New Files Remove Selected Files Time gaps between files Create atime gapof 1 seconds between each file Create the time gaps according to the file start times V Create Time Segments for file extents No time gaps between files Save Changes to omf File ox Cancel The mai
2. J Show Report v Delete All Units v Recalculate PCA Under most circumstances aligning around the Global Maximum or Global Minimum provides the best alignment of the waveforms Optionally all units can be deleted the Delete All Units checkbox and the PCA recalculated Recalculated PCA checkbox when the waveforms are realigned 140 Offline Sorter Offline Spike Sorting Software Preparation For Sorting After alignment around the global minimum as follows note how the waveforms are more tightly clustered in waveform space and principal component space 2500 my 800 usec By default unit assignments are ignored when performing alignment the Ignore unit assignments when aligning waveforms radio button is checked You can check the Align waveforms within each unit independently radio button after the units have been sorted This actually performs N independent alignment operations where N is the number of units For each alignment only the waveforms in a given unit are considered when choosing the alignment center for the operation This has the effect of adjusting timestamps to correct for triggering jitter CAUTION View reports with caution Use the Align waveforms within each unit independently setting with caution By its nature it appears to tighten up the clusters However this is a bit misleading because it was done with a priori knowledge of the clusters After
3. The information area includes information on license keys and a list of all the Plexon products and their licensed status on this PC If you have moved or added a key the Test the Key Again button provides a convenient tool to test license keys to ensure they func tion correctly Ifyou have more than one key installed the Next Key gt gt and lt lt Prev Key buttons appear You may use these buttons to cycle through and test all keys The code entry area is used to enter the unlock codes for optional programs and features If you have purchased optional items instructions for entering codes and testing keys are included with Plexon installation programs System Report The purpose of the System Report button is to help Plexon Support diagnose problems by listing system information Clicking the System Report button will first display a dialog box to allow you to display the system report on the monitor or save the report to a file that can be sent via E mail to Plexon Support Version 4 0 A 15 Appendix A Navigating the Plexon UI System Report Run a System Report on your computer display the results Run a System Report and save the results in a file After selecting a choice clicking OK will launch the standard Microsoft System Information tool The illustration below shows the System Information report displayed on the monitor 0 System Information File Edit View Tool
4. 00 255 eee core Som UNS NEX Ne csessarsunstneaineen a R a 255 5 6 10 Sort Delete All Companion SCAN Files cc cccceceeeeesneeceeeeeeeeaeeseeeeeeeaeeteaes 256 mod See rea ty ogie 25 ser E E E T E 256 218 Offline Sorter Offline Spike Sorting Software ST Crne NOUN os Sects ocr A ALEA 257 wda AUN ONT MUM S Kronna a EOSED 257 5 72 Unite Delete Selected UMi sicdscctisdieiaceseccgiemeelogtham ne t AA EA 258 5 73 Unis Delete All UNIS scciies socscctveseneneptcenditnedncaraesiterandosnel ranean ptandenierasaieenieeanieats 258 STA Unis ANN UNIS srin ANANN 258 ra UE Tona N a a 259 5 7 6 Units Invalidate All Waveforms in Selected Unit cccccceccseeeeeesesteeeeeseeaes 260 5 7 7 Units Add Waveforms to the Selected Unit cccccccccesseceeeesssneeeesessneeeeesenaaes 260 5 7 8 Units Remove Waveforms from the Selected Unit cccccssseeeeeeessteeeeeeeneaes 260 5 7 9 Units Remove All Except Selected Waveforms from Selected Unit 0 260 5 7 10 Units Automatically Recalculate Unit Templates after Each Change 260 5 7 11 Units Calculate Unit Templates from Existing Units 0 ececeeseeeeeeteeeeesteeeeees 260 5 7 12 Units Automatically Recalculate Fit Tolerances after Each Change 260 5 7 13 Units Calculate Fit Tolerances from Existing Units cceeeseceeeseeeeeeeeeeeees 261 Pee PONS Fe FU sy canta tassel nae ean pe
5. What File s to Store the Exported Data in File E PlexonData Plexon plx testwaveforms sorted mat Version 4 0 207 4 Spike Sorting Step by Step To export information 1 Select the Format of the export either text file MATLAB file if MATLAB is available on the computer or Microsoft Excel if available on the computer For text export choose which delimiter to use between values Delimiter choices include comma comma and space space or semicolon For Excel and text export check the Export a Row of Column Headers to get a line of header text that labels each of the columns This makes the exported file easier to read Select Which Channels to export If a current channel has been selected in Offline Sorter Software when the File Export Per Waveform Data menu selection was selected the default is the current channel only Also all channels can be exported to a single file For text and MATLAB exports another option is to do all channels but generate one exported file per channel In this case a channel number is appended to the filename Select What Waveforms to Export By default Offline Sorter Software exports all valid waveforms Check the Export Invalidated Waveforms to also include any waveforms that have been marked invalid in the exported information Uncheck Export Unsorted Waveforms to cause any waveforms that have not been assigned to a unit to be ignored during the export You can als
6. ISlHistMax ms 100 Use Range However you can override the default by selecting All in the Control Grid which forces all waveforms to be shown in the Clusters Views regardless of what is showing in the Waveforms view Controlling waveforms in the Timeline View by Units By default the type of waveforms shown in the Timeline view matches those shown in the Waveforms view because the Contin View parameter in the Control Grid defaults to As above However you can override the default by selecting All in the Control Grid which forces all waveforms to be shown in the Clusters views regardless of what is showing in the Waveforms view 4 6 1 2 Controlling waveforms viewed by Time Using the Timeline View The Control Displays checkbox in the upper left portion of the Timeline View detailed on page 48 can be used to restrict the waveforms shown in the Clusters and Waveforms views to those waveforms contained in the scroll region of the Timeline view You can use this feature to display animations of how things progressed during the course of the file for details see Section 5 3 View Menu on page 237 By selecting a Time Range The Waveforms Create Time Segments menu entry brings up a dialog box that allows one to enter a time range that is relative to the beginning of the data file or create any number of time segments within the file If the Waveforms View Only Selected Time Segments menu item is checked o
7. The title on the top contains the channel number and optionally the file name To change whether the file name shows up in the title select the Sort Summary Customization dialog entry in the right click menu on the Sorting Summary view or select the associated toolbar button The other entries on the form include e Filename the full path to the file Time amp Date the time and date that the data file was collected Whenever possible this time and date is obtained from the header records in the data file so from header appears on this line If the time and date cannot be obtained from the file header it is taken from the time date on the file itself in which case from file appears on this line e Wf Length the total length of the waveform in microseconds e Prethreshold the time between the start of the waveform and the threshold crossing in microseconds Version 4 0 85 3 Views and Displays e A D Freq the digitization rate in Hertz Number Of Units the number of units found on this channel e Sorted the time amp date that sorting was done Currently this is always the current date e Using the name of the algorithm used for the sorting e Features the features selected to define the features space All the sorting quality statistics depend on the choice of features e Multivariate ANOVA the string in parenthesis indicates whether or not unsorted waveforms are treated as a unit in the A
8. Close Project Import Markers All Commands Built in Menus New Menu Export to PLX v Export Markers e Categories This is a list of all the toolbar categories Select a toolbar category to see the buttons in the Commands area e Commands This area shows all the buttons and the associated menu commands that belong to the category you select You can select the command you want and drag it to the toolbar A 4 3 Keyboard Customization The Keyboard tab allows you to bind keystrokes to commands An illustration of the Keyboard tab follows Version 4 0 A 19 Appendix A Navigating the Plexon UI Customize Toolbars Commands Keyboard Run Menu Options Category v Commands i Key assignments A CtrleN Open Project Save Project Save Project As Close Project Import Markers Reset All Export Markers eat to PLX Press new shortcut key Export to NEX Description Create a new document e Category This is a list of all the main menu headings Select a menu heading to category to see the associated commands in the Commands area Commands This is a list of all the commands associated with the selected main menu heading in the Category area Key assignments This displays the current key assignment for the command selected in the Commands area e Press new shortcut key This allows you to enter a shortcut key combination for the com
9. References Webb Andrew Statistical Pattern Recognition Second Edition John Wiley amp Sons 2002 Shahani Maneesh Latent Variable Models for Neural Data Analysis Doctoral Thesis California Institute of Technology 1999 McLachlan and Peel Finite Mixture Models John Wiley amp Sons 2000 6 5 7 Valley Seeking Algorithm The Valley Seeking algorithm in Offline Sorter Software starts by calculating the number of neighbors that each point in feature space has A neighbor is defined as a point that is within a certain critical distance of the target point The critical distance is given by Dist 0 25 sigma Parzen Mult where sigma is the standard deviation of the distances of all points to the overall mean and the Parzen Mult is a user specified parameter so named for its similarity to the Parzen density estimation kernel Note Creating the neighbor counts inherently scales as N N where N is the number of points This fact limits the number of points that can be handled by this algorithm to several thousand before computing resources are overwhelmed The neighbor count for each point is then put into a list and the list is sorted so that the points with the most neighbors are at the top of the list 304 Offline Sorter Offline Spike Sorting Software Details of the Sorting Algorithms Next a set of seed points are found that are local maxima in that they have more neighbors than any of their neighbor
10. Version 4 0 69 3 Views and Displays sorted as Unit a and 40 are sorted as Unit b the color of the surface above that bin is 60 yellow by default blended with 40 green by default By default the surface view is displaying the data as a tower plot The tower plot shows a tower or elongated box for each bin where the height of the tower derives from the number of counts in the bin As shown in the following screenshot the Surface view can also display in a mode where the surface has a single vertex for each histogram bin The number of counts in the bin determines the height of the vertex Adjacent bins provide adjacent vertices that are connected together to form the surface To toggle the tower plot mode select Show Tower Plots from the right click shortcut menu or press the T key In addition to the surface formed by the contents of the Density Histogram the Surface view can also show another surface by fitting 2 dimensional Gaussians to the existing units The standard deviations of the points within the unit determine the size of the base of the Gaussian plot and the height of the Gaussian plot is proportional to the number of points in the unit To see the Unit Fits surfaces as shown in the following illustration select Show Gaussian Unit Fits from the right click shortcut menu 70 Offline Sorter Offline Spike Sorting Software Clusters vs Time View To return the display to Densit
11. indicates the number of spikes in the channel if the channel has only continuous data the number of spikes can be 0 Invalidated shows the number of invalidated waveforms in the channel Note By default this column is very narrow and it can appear to be blank To see the column information the column must be expanded by dragging the dividing lines on the header bar for the column Unsorted shows the number of spikes that have not been assigned to a unit Unit a z shows the number of spikes assigned to each unit Version 4 0 29 3 Views and Displays Note The currently selected channel is designated with an gt gt in the first column a See Section 6 1 Details of Channels and Sources on page 277 TIP Viewing Individual Channels Individual channels can be selected for viewing or sorting or both by double clicking the channel on the far left hand side of the page 3 2 3 Channel Parameters View The Channel Parameters view contains more detailed per channel information about the settings in effect Leg pee Fees Seka to in Zoom l aeeai SNA Lea Paes Cot TETSPK45 TETSPK49 TETSPK53 TETSPK5 TETSPK61 TETWBOI TETWBO5S TETWBOS TETWB13 TETWB17 TETWB21 TETWB25 TETWB29 TETWB33 TETWB3 TETWB41 TETRA 5PK 45 46 47 48 4915 250 pv 5PK 43 50 5152 24975 250 pV SPK 53 54 55 56 gt 74915 250 pV SPK 57 58 59 6
12. A 7 Appendix A Navigating the Plexon UI i Frame Marker Marker Occurrences aX e Docking a Window at the Application Frame When you begin to drag a window a transparent blue rectangle appears to indicate the position of the window and the four Docking Stickers appear individually at each edge of the application frame to allow you to dock the window to the respective edge To dock just move the mouse to the desired Docking Sticker and release the mouse button The docked window will extend along the entire length of the edge to which it is docked The illustration below shows all four screen Docking Stickers N 4 D Docking a Window at a Window Edge When you move the window inside another window the window Docking Stickers appear inside the window grouped together near the center of the window Releasing the mouse button while it is over one of these stickers except the center one will dock the moving window to the respective edge of the window associated with the window Docking Stickers The illustration below shows the window Docking Stickers Note that the shading of these Docking Stickers is different that the shading of the Docking Stickers in the previous illustration lt d 80 gt e Placing Windows into Tabs Releasing the mouse button over the center Docking Sticker will allow the moving window to occupy the same space as the window beneath and will create tabs along the bottom for switching the view
13. File PlexonData pl2 test files 4chDemoPL2 nex Invalidate unmatched waveforms Multiply Nex Timestamps by 1 Unsort unmatched waveforms x cme The operation works by matching timestamps from the NEX file to the waveform timestamps in the currently loaded file there is also an option that can be used to control what happens to the waveforms that cannot be matched to timestamp from the NEX file You can also multiply the timestamps from the NEX file by a factor to take into account the timestamp compression that can happen due to the NeuroExplorer 31 bit timestamp limit 5 6 10 Sort Delete All Companion SCAN Files Deletes all the companion SCAN files for the current channel See Section 4 8 5 Saving and Restoring Scan Results on page 214 for details 5 6 11 Sort Stop Sorting Stops the currently running sorting process 256 Offline Sorter Offline Spike Sorting Software Units Menu 5 7 Units Menu The Units menu contains entries for performing sorting operations on existing Select Unit s Delete Selected Unit Delete All Units Combine Units Swap Units Invalidate All Waveforms in Selected Unit Add Waveforms to the Selected Unit Remove Waveforms from the Selected Unit Remove All Except Selected Waveforms from the Selected Unit Automatically Recalculate Unit Templates after Each Change Calculate Unit Templates from Existing Units Automatically Recalculat
14. To select a filter click the arrow of the Low cut Filter droplist and select the filter type select the number of poles for the filter enter the cutoff frequency then press the Apply button t Ch 156 SPKC28 chavs Cont Ch 156 SPKC28 m Control Displays Center Selected Zoom 108 2 50 my Thr 10 43 w P sz Detect Low cut Filter Bessel 4 4 pole Cutoff Freq 250 Hz Apply Scroll Range 10 0000 0 0000 Zoom Out Zoom Note Changing the filter causes the histograms and the waveform displays to be redrawn In the Display tab of the View Options window if Auto Zoom is clicked the zoom also adjusts 118 Offline Sorter Offline Spike Sorting Software Waveform Acquisition From Continuously Recorded Data To apply the same filter to all channels instead of just the current channel from the Waveforms menu select Filter Continuous Data The Filter Continuous Data dialog box opens r Filter Continuous Data Which Channels Current Channel only All Channels in Continuous Source FILT_WB Chs 14 15 gt Filter Parameters Filter Type None 7 Low cut High cut Number of Poles 4 pole Cutoff Frequency 250 Hz To apply the same filter to all channels in a Source click All Channels in Continuous Source and select from the available continuous Sources Set the filter parameters and click Apply to app
15. 0 2372 0 2343 0 8725 0 8927 0 7842 0 7614 0 8093 1 0036 0 8994 0 9047 20 8509 0 9610 10 2613 1 1740 0 3712 0 1828 0 6601 0 3513 20 0525 0 0371 0 1697 0 0697 0 1217 0 0915 0 0730 0 0546 0 5368 0 2308 0 0663 0 3021 0 0523 0 1115 0 0911 0 2324 1 1305 2 4535 0 2372 0 2343 1 4770 0 8725 0 8927 0 8082 0 7842 0 7614 3 7855 0 8093 1 0036 0 9257 0 8994 0 9047 0 0727 20 8509 0 9610 0 5016 Pairwise Sort Quality __ Pairwise Sort Quality Statistics are not currently being calculated __ To enable check the Calculate unit pairwise statistics after each change in the Tools Options Stats page Features line In the top portion of the window the Features line shows which features were used for each dimension of the 2D and 3D feature spaces All sorting statistics are dependent on the feature space The Multivariate ANOVA section shows the p value and the F Statistics with the Hypothesis and Error degrees of freedom in parenthesis from a 2D and 3D Multivariate Analysis of Variance ANOVA on the sorted units in the currently displayed in 2D and 3D feature space e In 2D Cluster Space first row Parameters from Multivariate ANOVA test on clusters in 2D feature space e In 3D Cluster Space second row Parameters from Multivariate ANOVA test on clusters in 3D feature space Version 4 0 201 4 Spike Sorting Step by Step You
16. 246 Offline Sorter Offline Spike Sorting Software Waveforms Menu n Digital Referencing Apply the Referencing to All Channels in Continuous Source VJEnable wB Chs 65 68 Signal to use as the Reference An existing channel Source WB Chs 65 68 Channel 64 WBO1 CAR Common Average Referencing Channels to use to compute CAR CMR Source WB Chs 65 68 Use all channels in Source Channel Name The top portion of the display enables disables digital referencing and selects which continuous Source the referencing is applied to Note that all channels in this target Source will have the reference channel subtracted from them with one exception if a single existing channel is used as the reference channel and that channel is in the same Source that the reference is being applied to then that reference channel within the Source does not have the referencing applied to it as it would result in data values of strictly 0 for the entire channel The Enable checkbox turns digital referencing on or off for the continuous Source selected in the droplist Note that each continuous Source remembers whether digital referencing has been enabled to it independently of all other Sources With multiple invocations of the Digital Referencing dialog it is possible to set up digital referencing on several continuous Sources with each Source using a different r
17. PL2 have a concept of a Source which is a collection of related raw channels Each Source has a unique identifier number Raw channel numbers only have to be unique within each Source However the combination of Source identifier and raw channel number together provide a unique identifier for each channel of data within a file Internally Offline Sorter Software uses this two level scheme of a Source identifier together with the raw channel number to uniquely identify each raw channel It is perhaps easiest just to think of Sources as collections of similar raw channels All raw channels within a Source must share certain characteristics they must all be of the same data type either Spike or Continuous they must all have the same digitization frequency the same number of ADC bits and the same maximum ADC voltage For spike Sources all spikes within all the channels within the same Source must have the same number of data points in them referred to as NPW Number of Points per Waveform Also all channels with a Source must have the same trodality they must all be single electrode stereotrode or tetrode channels See Section 3 2 1 Sources View on page 27 for a description of how Offline Sorter Software displays information about the currently loaded Sources Other facts about Sources e Each Source has a name For formats other than PL2 the Source name is heuristically generated e A Source may also contain
18. Reload Channel from File 237 Edit Previous Channel in History Next Channel in History 237 View Menu 237 List of View Menu Options 237 View Animate 238 Select Menu 240 Select Next Channel Previous Channel 240 Select Next Unit Previous Unit 240 Select Next Waveform Previous Waveform 240 Select Next Segment Previous Segment 241 Select Feature Space Definitions 241 Waveforms Menu 241 Waveforms View Only Currently Selected Unit s 242 Waveforms View Only Unsorted Waveforms 243 Waveforms View Only Selected Time Segments 243 Waveforms Create Time Segments 243 Waveforms Use Only Currently Visible Waveforms 245 Waveforms Use Only Unsorted Waveforms 246 Version 4 0 xiii xiv Waveforms Invalidate Waveforms 246 Waveforms Invalidate All Except Selected Waveforms 246 Waveforms Mark All Waveforms As Valid 246 Waveforms Invalidate All Waveforms in Selected Unit 246 Waveforms Perform Digital Referencing on Continuous Data 246 Waveforms Up sample Continuous Data 248 Waveforms Filter Continuous Data 248 Waveforms Detect 249 Waveforms Align 250 Waveforms Re Threshold 250 Waveforms Remove Short ISI Waveforms 250 Waveforms Auto Zoom Waveform Displays 250 Sort Menu 251 Sort Change Sort Method 251 Sort Perform Automatic Sorting 251 Sort Perform Template Sorting 253 Sort Apply Template Sorting with Current Templates 254 Sort Apply Template Sorting wi
19. The Semi Automatic and Automatic sorting algorithms behave differently depending on how they are initiated e If the sorting is initiated from the 2D Clusters view the Clusters vs Time view or the Surface view e g by pressing the Find Units button on the view specific toolbar the sorting is done in 2D feature space Version 4 0 299 6 Reference Topics e If the sorting is initiated from the 3D Clusters view the sorting is done in 3D feature space e If the sorting is initiated from the Sort Perform Automatic Sorting main menu item the radio buttons in the Sort In box control whether the sorting is done in 2D or 3D feature space 6 5 2 Template Algorithm The Template matching sorting algorithm is designed to mimic the online real time sorting performed by the Plexon MAP data acquisition hardware For each waveform the algorithm 1 calculates the sum of squares differences from this waveform to all the templates 2 finds the template with the minimum sum of squares difference 3 if the minimum difference is less than the Fit Tolerance for this template the waveform is assigned to this template s unit You can adjust the Fit Tolerance measured in units of 0 01 of the maximum A D value in the Units view Refr ISI fish Note For Plexon MAP users The MAP system hardware uses a slightly different scaling factor on the Fit Tolerance To use Fit Tolerance values that will give identical behavior in Offli
20. after sorting runs the Remove Outliers tool in either 2D or 3D feature space using the currently set Outlier Threshold RemoveShortISI runs the Remove Short ISI tool using the currently set Refractory Period AssignUnsorted2d AssignUnsorted3d runs the Assign Unsorted tool in either 2D or 3D feature space using the currently set Outlier Threshold KeepWfsBetweenEvents runs an interval filter that keeps only the waveforms between two specified events with an optional time offset from each event KeepWfsInSpan runs an interval filter that keeps only the waveforms within a time range around a specified event KeepWfsOutsideSpan runs an interval filter that keeps only the waveforms that are outside of a time range around a specified event KeepWfsBetweenTimes runs an interval filter that keeps only the waveforms within a specified time range InvalidateWfsBetweenTimes runs an interval filter that invalidates the waveforms within a specified time range SortSummaryToPP after sorting dumps the sort summary to PowerPoint for each channel LogStats2d LogStats3d after sorting dumps the 2D or 3D sort quality stats to the log file 316 Offline Sorter Offline Spike Sorting Software Batch File Processing Scan performs a parameter scan as configured with the Set ScanXXX commands SaveScanResults saves the results of a previous ForEachChannel Scan command to a file that can be la
21. on page 249 The goal is to calculate the standard deviation of just the noise without any spike signal present However the very definition of what is signal and what is noise depends on establishing a threshold signal crosses the threshold but noise does not So the approach used in Offline Sorter Software is to do a preliminary fake thresholding of the data just to make a preliminary classification of which samples are part of a signal and which are part of the noise 1 The standard deviation of the full signal is calculated from the sample values again using only the first buffer of continuous data Call this the full signal sigma Version 4 0 75 3 Views and Displays 2 Then a fake positive and negative threshold is applied to the raw data at 2 7 times the full signal standard deviation calculated in Step 1 Any sample that is between 2 7 full signal sigma lt sample lt 2 7 full signal sigma is considered to be noise it did not cross either the positive of negative threshold and any sample less than 2 7 full signal sigma or greater than 2 7 full signal sigma is discarded 3 Find the noise sigma by calculating the standard deviation of just the samples classified as noise by the step 2 procedure Again this procedure should be considered as a method to get a better estimate of the standard deviation of just noise by using the above procedure to reject signal from the calculation The
22. Note that the default layout depicted above does not include all of the views that are available in Offline Sorter Software 18 Offline Sorter Offline Spike Sorting Software Plexon Inc Chapter 3 Views and Displays eM PUG oiia 21 22 Mio Gnid VONT si iashsnendicatvandeainieiserenadeennitiesi redad sania 25 3 2 1 NOES VIEW corssccssiasixstiuandviossioiinesteianmadssetuadetonstuies 27 3 2 2 Channels VIEW rssicon iniii 29 3 2 3 Channel Parameters VieW cccsccccceessseceeeessteeeeeseees 30 3a File Summary VEW mesnin a 32 3 2 65 lime Segments VIEW ss6 cccviccseiaccivsagnedeceisessdnootaceseronteeder 34 9 2 0 Gort Datalle VIEW rsisi aen n ia 35 a ENOS VON aaan a aA 36 S2 o SNS VION arire aE i 36 Tao PFOA VION onra 36 niemo k e E E E E E E A E N E 36 Soe AT Templates Vew lies stra dacetiuasduekesedvaderesaddncunesdnsuudeesdsaulous ETA 3 2 12 Adaptive Templates History View 37 3 213 Channel Map VIEW oriras aa 37 rie AE Messages Vei onsnaisie ppdasaatdassstantvad potaiia 37 cs OTO T i 38 SUIS VON crannan an en aR 42 S0 Waveldime WOW asrina iaa iaaa 45 LO EIS VIEN aripa kaaa aaa kaaa ESSA 48 3 6 1 Stripchart Display in the Timeline View ceceee 48 3 6 2 Other Features of the Timeline View ccccsseeeeee 51 3 7 2D and 3D Clusters VR WS wsicsiisiieisiedssovsiierd savvviedioninnm ea oainninn 52 3 7 1 Displaying 2D and 3D Clusters Views ccceeee 53 3 7 2 Clusters Views Common Function
23. Previous Channel To move quickly to the next or previous channel in the list of channels click arp Select Next Channel or Select Previous Channel The toolbar buttons can also be used 5 4 2 Select Next Unit Previous Unit Offline Sorter Software has the concept of a currently selected Unit within a channel This function will change the current unit to the next or previous unit The amp u 2 toolbar buttons can also be used 5 4 3 Select Next Waveform Previous Waveform Offline Sorter Software has the concept of a currently selected Waveform within a channel This function will change the current waveform to the next or previous oh waveform The toolbar buttons can also be used 240 Offline Sorter Offline Spike Sorting Software Waveforms Menu 5 4 4 Select Next Segment Previous Segment Offline Sorter Software has the concept of a currently selected Time Segment This function will change the current Time Segment to the next or previous Time apy Segment The toolbar buttons can also be used 5 4 5 Select Feature Space Definitions Offline Sorter Software can remember up to three feature space definitions that are commonly used and it enables you quickly to put those pre defined feature spaces into effect For example if you are constantly switching between a PC1 PC2 PC3 feature space and a Slice 1 Peak Valley feature space Feature Space 1 can be defined to be
24. and a high cut filtered FP channel to the data file 282 Offline Sorter Offline Spike Sorting Software Details of Channels and Sources In general Offline Sorter Software is unaware of the fact that multiple raw channels may have originated from the same physical electrode Thus if you want to view both the spikes and some flavor of continuous data that came from the same physical electrode together in the Timeline view the user must explicitly tell Offline Sorter Software which channels to view Note The Timeline view in Offline Sorter Software can only view a spike channel together with a continuous channel viewing two spike channels together or two continuous channels together is not possible There are multiple ways to accomplish viewing related or even unrelated spike and continuous channels together the Timeline view in Offline Sorter Software Selecting an Offline Sorter Channel will always reset the Timeline view to show the Source s associated with that channel Suppose using the above example data file the user loads Offline Sorter Channel 3 The Sources view shows the corresponding SPK Source marked with the S gt for spike indicator Name Te Chs Toda Frea Spike 1 64 1 Cont 65 128 1 Cont 129 192 1 Cont 193 256 1 2500 0000 7 8 ia z 2500 0000 6128 Y i 6 2500 0000 129 192 Y 2500 0000 a Version 4 0 283 6 Reference Topics Suppose t
25. assign the waveforms to an optimal number of clusters For more details see Section 6 5 7 Valley Seeking Algorithm on page 304 The Valley Seeking algorithm operates differently depending on which view initiates it and which options have been set see Section 6 5 1 Feature Space Used for Sorting on page 299 Note The Valley Seeking algorithm scales as N N where N is the number of points So for large number of points more than a few thousand the algorithm can use unreasonable amounts of memory and take a long time to complete Offline Sorter Software can limit the number of points that are used as input to the Valley Seeking algorithm The Sort tab on the Tools Options dialog box contains the Limit Number of Waveforms used in Valley Seeking sorting checkbox that when checked limits the number of waveforms sorted to be the number in the associated edit box Offline Sorter Software then uses every nth waveform in the Valley Seeking calculation where n is the smallest integer such that the total number of waveforms used is less than the specified maximum After the Valley Seeking algorithm sorts the waveforms you can optionally operate on the unsorted waveforms those that were skipped by using the Assign Unsorted Waveforms tool This is the same tool that is available from the Tools Assign Unsorted to Closest Unit menu selection See Section 5 8 9 Tools Assign Unsorted to Closest Unit on page 267 You can use the Assign
26. display shows waveform segments positioned according to their timestamps If there is no continuous data present for the channel being viewed then this waveform segments portion will fill the entire stripchart display vertically Colors represent the unit assigned to each waveform segment Individual waveforms can be selected for viewing in the Waveforms view and in the Waveform Inspection view by clicking on the waveform with the left mouse button e Continuous stripchart display The bottom portion of the stripchart display is only present when there is continuous data for the channel If there is no spike data selected to be shown then this waveform segments portion will fill the entire stripchart display vertically The voltage versus time trace of the continuous data is plotted The voltage versus time trace of the continuous data is plotted Version 4 0 49 3 Views and Displays Control Displays This checkbox restricts waveforms displayed in the Waveforms view and Clusters view to those contained in the Scroll range setting of the Timeline view Center Selected If checked when a waveform is selected using the method described in Section 3 7 2D and 3D Clusters Views on page 52 it causes the time range displayed to change such that the selected waveform is shown in the center of the window Zoom This item represents the zoom factor for the voltage display which is the same as the Zoom parameter in the Co
27. drag the mouse to the toolbar area and place the menu as a toolbar there The illustrations below show highlighting the Tear off Menu Handle to begin dragging the toolbar dragging the toolbar across the screen and finally docking the toolbar in place Only some Plexon applications support tear off menus fF Define New Marker p Edit Selected Marker DAES PIRI PPA PARA Fafa pafa D Ds Re Run j KP eRe te A O Rid E Time OE 03000 37000 4 GB MOA nF fara fafa EFRI P ae ee Offline Sorter Offline Spike Sorting Software A 2 2 Toolbars Each toolbar has a dropdown button at the right that will allow you to add or remove buttons from toolbars and will also allow you to access the Customize dialog box When you click the dropdown button the Add or Remove Buttons dropdown menu will appear as shown below 1X 1X R Add or Remove Buttons 7 You will see that the dropdown menu also has a dropdown button If you click that button you will see a menu list of the available toolbars and a menu command to allow access to the Customize dialog box Expanding one of the toolbar menus will display all of the available toolbar buttons for that particular toolbar and a checkbox beside each one that will indicate whether or not the button is visible on the toolbar There is also an option to reset the toolbar back to its factory default setting Reset Toolbar Ver
28. is the unsorted waveforms on the sig002 channel By default any Events that are selected to show by clicking Show in the Events view of Info Grid Views are plotted each on its own raster lines in the color selected in the Events view The rasters for Events can be shown either on the top default or on the bottom of the spike timelines by checking or unchecking Show Events on Top most Rasters the context menu Alternatively select Show Events Across All Rasters in the right click context menu to plot the Events as vertical lines that cross all the raster lines Also you can select Show All Events in the context menu which is equivalent to clicking Show for all Events Selecting Show Time Segments Boundaries in the right click menu will plot vertical lines at the time positions where time segments begin and end and selecting Zoom Time Range To Selected Segment will zoom and scroll the time range so that the currently selected time segment fills the time range 3 19 Scan Graph View The Scan Graph view displays a graphical view of the scan results which are shown in tabular form by the Scan view For more information see Section 4 5 12 Parameter Scans on page 179 To display the Scan Graph view Select the View Scan Graph menu command or press the button on the toolbar 94 Offline Sorter Offline Spike Sorting Software Scan Graph View The Scan Graph view shows a simple graph of some selected sort
29. s software products Included are e CinePlex Studio e CinePlex Editor e MAP2 e Offline Sorter Software This appendix covers common functionality of the look and feel between different Plexon software applications The discussion includes the following sections e Screen Elements e Standard Menu Items and Dialogs e Customization A 2 Offline Sorter Offline Spike Sorting Software A 2 Screen Elements A 2 1 Menus The menu bar of the application contains the names of all of the menus for the application Each menu name has a letter underlined which indicates the hot key combination for that menu To activate a particular menu you may click the left mouse button on the name or press and hold the ALT key while pressing the underlined letter The illustration below is the main menu of CinePlex Editor Ble Edt View Animate Looping Find Tools Window Run Help Each menu of the Plexon User Interface contain menu commands and may also contain Icons Hot Keys and a Tear off Menu Handle for some applications as shown by the illustration below of the Edit menu of CinePlex Editor Menu Commands Tear off Handle Hot Key Adit Selected Marker pelete Selected Marker Copy Selected Marker I Add New Marker Occurrence Edit Selected Marker Occurre Delete Selected Marker Occurr H Nelete All Marker Occurrenceg eystroke Inserts Marker Nn Press Inserts Marye While all Plexon applications
30. the cluster centers are evenly spaced along a diagonal of the bounding box Std E M Scan is very similar to the K Means scan in that it scans over the number of units and the initial cluster centers are placed according to the Seed cluster pattern but the Std E M sorting algorithm is run at each step Valley Seeking Scan Scans over a range of Parzen Multipliers The range is specified in the Tools Options Sort tab as is the step increment value T Dist E M Scan Scans over a range of D O F Multipliers The range is specified in the Tools Options Sort tab as is the step increment value Note that the running one of these scanning sorting methods actually runs the same parameter scan that can be done using the Scan view In fact running one Version 4 0 183 4 Spike Sorting Step by Step of these sorting methods will populate the Scan view and the Scan Graph view see Section 4 5 12 Parameter Scans on page 179 with the results of the scan 4 6 Unit Cleaning 4 6 1 Controlling Waveforms Displayed To inspect the sorting more closely it is sometimes desirable to select which waveforms are displayed in the windows There are two main ways of limiting the waveforms that display units and time interval 4 6 1 1 Controlling waveforms viewed by Units Offline Sorter Software can display the waveforms from only selected unit s in the Waveforms view and in the Clusters views Controlling waveforms in the W
31. 00 mi D Thr 31 0 1552 p ae Detect Waveforms Options Up Sampling None Frequency Low cut Filter None Scroll Range 10 0000 0 0000 Zoom Duk Zoomin lt 4 As before the threshold in any view can be adjusted by clicking and dragging the threshold line which is the red line in each of the preceding traces By default Offline Sorter Software constrains the threshold level to be the same on all electrodes within the stereotrode tetrode When the threshold is adjusted on one electrode the threshold changes for all electrodes However to adjust the threshold for each electrode independently click Detector and Waveform Options and in the Waveform Detection Options dialog box click For stereotrode tetrode data allow an independent threshold for each electrode For waveform detection on stereotrode or tetrode continuous data a threshold crossing on any electrode results in the capture of the waveform across all electrodes As in the single electrode case if the Detector is specified to be anything other than Raw Signal each of the traces again divide into two halves showing both the raw signal and quantity used for thresholding Version 4 0 79 3 Views and Displays 3 12 Continuous Frequency Spectrum View If Continuous Data has been loaded into Offline Sorter Software the software can perform an FFT on the continuous data and display a frequency spectrum of the signal This is not directly relevant to Off
32. 1 f Use Dual Thresholds Always set Thr 2 Thr E Continuously Sample Raw Signal Adjust Gains Alignment During Detection Raw Signal Only Detect Now _ Enable After threshold crossing align to the largest peak Same Side as Threshold Either Side Cancel The Waveform Options window contains the following items Total Waveform Length This item specifies the total length in psec and number of samples of the thresholded waveforms that are extracted from the continuous data Prethreshold This item specifies the time in usec and number of samples prior the threshold crossing included in the extracted waveforms Dead Time This item specifies the minimum time in usec and number of samples between extracted waveform segments For stereotrode tetrode data This item when checked allows independent threshold setting for each electrode By default a single threshold value is used for all electrodes Detector The item indicates quantity used for thresholding either Raw Signal Energy of the Signal or Nonlinear Energy of the Signal Energy Window Width This item specifies the width of the moving win dow over which the quantity is averaged for energy and nonlinear energy detector options only Version 4 0 121 4 Spike Sorting Step by Step e Use Dual Thresholds This checkbox enables setting two different thresh olds with the waveform segments being taken if the
33. 36 Templates View 37 Adaptive Templates History View 37 Channel Map View 37 Messages View 37 Control Grid 38 Units View 42 Waveforms View 45 Timeline View 48 Stripchart Display in the Timeline View 48 Other Features of the Timeline View 51 2D and 3D Clusters Views 52 Displaying 2D and 3D Clusters Views 53 Clusters Views Common Functionality 54 Axis Feature Selection 54 native loaderWaveform Highlighting 56 Controlling Which Waveforms Are Displayed 56 Short ISI Spike Pairs Display 57 Cluster Positioning 57 Per View Toolbars Hints and Statistics Display 58 Colors 59 Point Size 59 Automatic Cluster Redraw 59 Zooming and Panning 60 Context Menus 60 2D Clusters View Details 61 3D Clusters View Details 65 Surface View 69 Clusters vs Time View 71 Waveform Inspection View 73 Waveform Detection View 74 Threshold Calculation 75 Adjusting the Threshold for Waveform Detection 76 Changing the Detector 76 Stereotrode and Tetrode Waveform Detection 78 Continuous Frequency Spectrum View 80 Xx Offline Sorter Offline Spike Sorting Software Offline Sorter Offline Spike Sorting Software Feature vs Feature View 80 Sorting Summary View 84 Customizing the Sorting Summary 87 Output to PowerPoint 88 Outputting for All Channels 88 PCA Results View 88 ISI Histograms View 90 Cross Correlograms View 91 Rasters View 92 Scan Graph View 94 Sort Quality vs Time Segment Graph View 97 Threshold Scan Graph 97 Overlaps Analysis View 99 Overlap
34. Add Wfs and Remove Wfs Offline Sorter Software shows the multiple secondary units with a dashed box highlighting the unit template but with the default background color When the Waveforms view setting in the Control Grid is set to Sel Units s Offline Sorter Software also displays the waveforms for the multiple selections in the Waveforms view Clusters views and Timeline view This is a convenient way to compare and contrast units Also certain operations like Units Combine Units and Swap Units use multiple unit selections To select multiple units follow the Windows standard for selecting multiple items e To select a unit click the unit e To select non adjacent units press and hold down CTRL and then click each unit e To select adjacent units click the first unit press and hold down SHIFT and then click the last unit e To cancel an existing multiple selection click any unit Units can also be selected by using the Units Select Unit s dialog box Version 4 0 43 3 Views and Displays r Select Multiple Units Unsorted Unit b Unit c Caneel The right click menu on the Units view provides easy access to some common functionality that is available from other main menu selections such as the ability to delete the selected unit delete all units swap units combine units invalidate all waveforms in the selected unit select multiple units using a dialog and to re so
35. Automatic Clustering Methods 143 4 5 2 Manual Clustering using the Contours Method 146 4 5 3 Manual Clustering using the Waveform Crossing Method 153 4 5 4 Semi Automatic Clustering using Templates ceseeeeeeee 157 Version 4 0 107 4 Spike Sorting Step by Step 4 5 5 Manual Clustering using the Boxes Method n 163 4 5 6 Manual Clustering using the Lines Method ccccceeeeeeerees 165 4 5 7 Manual Clustering using the Bands Method cceeteeeeerees 168 4 5 8 Semi Automatic Clustering using K Means ccecceeeeeeeeeeneees 171 4 5 9 Semi Automatic Clustering using Standard E M ceeeeeeees 175 4 5 10 Automatic Clustering using Valley Seeking Method 0 176 4 5 11 Automatic Clustering using T Distribution E M Method 178 4S 1 Parameter SEAE a tetera anaemia anes 179 4 5 13 Automatic Clustering Using the Scanning Methods 00 182 LE UNE ORANIN Deeper peeerrer rerenere nr petsere nrprnce rt reer cree rrererr tert eree Terr tres Tree 184 4 6 1 Controlling Waveforms Displayed sscceceseeeseseeeeeeeeeeeeeeeeees 184 4 6 2 Waveform Selection Op oNS sarsii 186 46 3 Cleaning the SOOO TION rannsaki 187 4 6 4 Removing Spikes with Short Inter spike Intervals 0cee 190 4 6 5 Rescuing Overlapping Waveforms ccccecceeeeseeeeeeeeeeeteeeesees 194 dl Examining De SONN si vs is us iaoa
36. Clusters View Displays droplist on the Tools Options Stats tab The value of that statistic appears along with the percentage change from its previous value that the last sorting related action caused If the action made the clustering better Version 4 0 203 4 Spike Sorting Step by Step according to the statistic the bar is green if the sorting quality decreased the bar is red ne E X amp d Unit Find Units Delete Unit Add i Ad p 2 01917e 031 J3 11 PsF 11114 8 DB 0 309 Dn 1 62 4 7 3 Examining the Sorting vs Time It is advisable to examine the time structure of the data using the Clusters Vs Time view see Section 3 9 Clusters vs Time View on page 71 This can reveal situations such as the waveforms occurring in bursts units appearing or disappearing etc 204 Offline Sorter Offline Spike Sorting Software Saving the Sorting Results 4 8 Saving the Sorting Results 4 8 1 Saving Timestamps and Waveforms If Offline Sorter Software is used to resort spikes from a file with previously sorted waveform spike data e g a Plexon PLX or PL2 data file Datawave Cybernetics NEV or CED Spike 2 the resorted waveforms and timestamps can be saved into another file of the same format All records in the original file are preserved in the output file except that the unit assignments for spike records are modified to reflect the sorting that has been performed in Offline Sorter Software
37. File C PlexonData Plexon plx ev024 plx Process The behavior of the example file is to e Set the feature space to be PC1 and PC2 e Set the parzen radius multiplier to 1 0 e Open chan9threeunits3 plx For each channel e Do 2D Valley Seeking sort using PC1 and PC2 e Send the sort summary to PowerPoint e Log the 2D sort quality stats to the log file Version 4 0 321 6 Reference Topics Save the timestamps to chan9threeunits3 nex Open ev024 plx For each channel e Do 2D Valley Seeking sort using PC1 and PC2 e Send the sort summary to PowerPoint e Log the 2D sort quality stats to the log file Save the timestamps to ev024 nex Set the parzen radius multiplier to 2 0 Do the same thing as described above to both files but this time using a parzen multiplier of 2 0 6 9 3 Notes Executing a Set command via a batch file will generally affect a parameter that can also be set via the Offline Sorter graphical user interface However if a Set command changes a parameter the new value of that parameter will only be in effect for the duration of the batch file execution The new value will not be in effect the next time Offline Sorter Software is started Any values not explicitly set by the batch commands obtain their values via the settings that were in effect the last time that Offline Sorter Software was run Note that some parameters that affect batch operations can ONLY be inherited in this fashion s
38. Likewise if alignment was performed on the spikes Offline Sorter Software saves the alignment parameters The Sort Details view of the Info tab shows what sorting has been performed on which channels using which sorting parameters and it is a good indication of what will be written to the TPL file To save the information in a TPL file 1 Sort each channel using templates see Section 6 5 2 Template Algorithm on page 300 adjust template tolerances set the sort width etc or using the Contours method 2 Select File Save Sort Info to tpl menu command The TPL file contains information only for the channels that have been sorted Note The only sorting methods that can be captured in a TPL file are Template and Contours If a channel was sorted using another method e g Valley Seeking you can approximately capture the results into a TPL file as a template sort To do this one needs to have Offline Sorter Software adjust the unit templates and fit to frames such that the results of the template sort approximate the desired Version 4 0 213 4 Spike Sorting Step by Step AN sorting For more information see Section 4 5 4 1 Managing Unit Templates and Fit Tolerances on page 158 CAUTION Uncaptured Operations You can perform sequences of operations on a channel that cannot be captured in a TPL file Thus the same operations are not exactly duplicated when a saved TPL file is re applied Examples of o
39. M Method has a setting for the number of degrees of freedom DOF for the fitting procedures Use of the theoretically predicted value of this parameter results in poor sorting The D O F Mult value multiplies the theoretical DOF setting Smaller values of this multiplier generally result in slow convergence to a larger number of clusters larger values result in rapid convergence to a smaller number of clusters The Semi Automatic Clustering using Standard E M algorithm depends upon an annealing rate parameter Beta When Beta is 1 a completely standard E M algorithm runs When the value is other than one a variant of the standard E M algorithm runs Waveform View specifies which waveforms Offline Sorter Software draws in the Waveforms view either All waveforms Selected Unit s Unsorted or only the Selected Waveform Selected Unit s permits a quick selection of units by clicking in the Units view Clusters Views specifies which waveforms appear in the Clusters views either All waveforms or Same as above which displays the waveforms specified in the Waveforms view parameter Timeline View specifies which waveforms are shown in the Timeline view either All waveforms or Same as above which displays the waveforms specified in the Waveform View parameter Only Segment is an option that displays the waveforms for the currently selected time segment only See Section 3 2 5 Time Segments View on page 34 Th
40. MAP files is available only via the Neuroshare DLL which can be obtained from www neuroshare org One must download this DLL file and use the Alpha Omega tab of the Tools Data Import Options dialog box to set the name and location of this DLL As with most Neuroshare DLLs there are some issues with handling gains for Alpha Omega files the voltages displayed in Offline Sorter Software may not be accurate There is also an indication as of the time of this writing that units beyond Unit a are mislabeled 5 1 2 2 CED Spike 2 Import for CED Spike 2 SMR files is available via CED s Neuroshare DLL To import the data via the Neuroshare DLL one must download the Neuroshare DLL from www neuroshare org and use the CED Spike 2 tab on the Tools Data Import Options dialog box to set the name and location of this DLL CED Spike 2 files that have a different number of samples per spike waveforms on different channels do not load properly into Offline Sorter Software all spike channels must have the same number of samples per spike waveforms Only single electrode data types are supported by the native loader 5 1 2 3 Cyberkinetics Import for Cyberkinetics also known as Black Rock Microsystems formerly Bionic Systems NEV files is available only via Cyberkinetics Neuroshare DLL To import the data with the Neuroshare DLL one must download this DLL file see www neuroshare org and use the Cyberkinetics tab in the Tools Data Import
41. Options dialog box to set the name and location of this DLL Units marked with a 1 unit code Noise are imported as invalidated spikes 5 1 2 4 Data Translations Import for Data Translations DCF files is available via a native loader This format contains only continuous data samples 5 1 2 5 Datawave Import for Datawave UFF and Experimenters Workbench EWB files are available via a native loader The Non EWB format supports all data types for spikes continuous data and events the EWB format supports all data types for spikes and events For continuous data there is a mismatch between the continuous sampling frequency reported in the header and what is actually seen in the data It leads to gaps in the continuous data trace 222 Offline Sorter Offline Spike Sorting Software File Menu 5 1 2 6 MultiChannel Systems Import for MultiChannel Systems MCD files is available via a native loader and via the Neuroshare DLL which can be obtained from www neuroshare org You can toggle between using the native loader or the Neuroshare loader using the MultiChannel Systems tab of the Tools Data Import Options dialog box To use Neuroshare to load MultiChannel Systems files one must download this DLL file and use the MultiChannel Systems tab of the Tools Data Import Options dialog box to set the name and location of this DLL in the Neuroshare DLL Location section Support for MultiChannel Systems files depends on
42. PC1 PC2 PC3 and Feature Space 2 to be Slice 1 Peak Valley by using the User Defined Feature Spaces section of the Feature Calc tab of the Tools Option window Then you can use the toolbar buttons corresponding to each of the three user defined feature spaces or the menu items under Select Feature Space Definitions which can be bound to keystrokes for faster selection to flip between the feature spaces without having to use the toolbar feature drop lists The User Defined Feature Spaces dialog provides settings in all three dimensions of a feature space for 3D feature spaces If a 2D feature space is used or displayed only the first two dimensions are used User Defined Feature Spaces Feature Space 1 Assign Current Feature Space x PC 1 y Pc2 z rca Feature Space2 Assign Current Feature Space x Slice 1 yl y Slice 2 v z Energy x Feature Space 3 Assign Current Feature Space x Peak y valey Z Timestamp x To define a user defined feature space use the drop lists to select the feature for each dimension Or if the Clusters view is already showing the desired feature space click Assign Current Feature Space 5 5 Waveforms Menu The Waveforms menu contains entries for performing various operations on Waveforms other than Sorting them see the Sort menu Generally these operations are performed before sorting the spikes Following is an image of the Waveforms menu Ve
43. Print Click Print to print a copy of the Quick Reference About Clicking the About item displays the About dialog box The text of the About item varies according to the application The About dialog contains the version number and build data of the application links to the Plexon website and support e mail and buttons for Licensing System Report and Manage File Extensions About CinePlex Editor Application CinePlex Editor Application Yersion 2 0 0 Built on Mar 25 2009 Copyright C 2005 2008 Plexon Inc Visit our web site www plexoninc com Support e mail support plexoninc com System Report Manage File Extensions e Licensing Clicking the Licensing button displays the Plexon License Management dialog box The Plexon License Management window includes the complete licensing information for Plexon products The A 14 Offline Sorter Offline Spike Sorting Software window includes the following three areas information key testing and code entry Plexon License Management Sentinel Versions Library 6 4 Driver 7 4 0 Key found Key Serial Number 02156 Customer ID 00423 Key Number 00001 Products Licensed Offline Sorter YES WaveTracker MES Recorder NI YES Recorder ADS Wee Rasputin YES 128 channels NeuroSurgery WS MES MEA WS YES CinePlex 2 YES with Tracking Plexus MES Test the Key Again Unlock Additional Programs and Features 1 2
44. ScanvalleySeeKking Of encaissat anaoa a a 325 CIOS FEROCE O ea 325 CIG Save TONER O ruinie a a a EE E a AEN AAEN 325 T Lace Fio Up aaa a 325 Pe Wl Ba PLZ MOS orraa recy a E AEE 325 6 11 2 Stasies Callana scone EE 326 DRAE EA A A E A A rere E E err eree yer ree ay 326 6 11 4 Ignoring Continuous Data Channels cccccccceceeeeeeeeeeeteeeeeeeeeseaaeseeeneees 326 Ue Na PCA ATE ccc ars seen casas eset ppcrda cues a E aa eonetaieanen 327 276 Offline Sorter Offline Spike Sorting Software Details of Channels and Sources 6 1 Details of Channels and Sources While using Offline Sorter Software it is important to keep in mind how the Offline Sorter Software defines and treats Channels and Sources 6 1 1 Raw Channels and Sources There is a distinction between Offline Sorter Channels and raw channels that are read in from data files Define a raw channel to be a channel as represented in a data file A raw channel might contain spike data or continuous data but not both Sometimes a raw channel of spikes and a raw channel of continuous data were collected from the same physical electrode but these are still represented in the data file as separate though perhaps somehow associated raw channels A raw channel from a data file always has a raw channel number to identify it However the number associated with a raw channel might not be unique throughout the entire data file some file formats e g Plexon
45. See Section 5 8 5 Tools Re Threshold Waveforms on page 263 5 5 17 Waveforms Remove Short ISI Waveforms Select this menu item to automatically invalidate spikes that are unreasonably close to other spikes in time See Section 4 6 4 1 Automatic Short ISI Spike Removal on page 191 5 5 18 Waveforms Auto Zoom Waveform Displays Applies the auto zoom procedure described in Section 4 4 1 1 Waveforms View on page 127 to set the zoom in the Waveforms and Timeline views 250 Offline Sorter Offline Spike Sorting Software Sort Menu 5 6 Sort Menu The Sort menu contains entries for performing sorting operations on existing waveforms Following is an image of the Sort menu Units Tools Window Run Help Change Sort Method Perform Automatic Sorting Perform Template Sorting Apply Template Sorting with Current Templates Apply Template Sorting with Adaptive Templates Adaptive Templates Settings Save Sorting to SORT File Restore Sorting from SORT File Save Sort Info to TPL File Sort Using TPL File Sort Using NEX File Delete All Companion SCAN Files The sections that follow explain the contents of each Sort menu options 5 6 1 Sort Change Sort Method These are checkable menu items that show the currently selected sort method and allow changing the current sort method The current sort method will be KA applied when you press the Find Units button in th
46. Short ISI Spike Pairs Display The Clusters views show the waveforms assigned to the same unit with interspike intervals less than the Refractory ISI inter spike interval by drawing a line between the two corresponding dots in feature space You can turn on or turn off the drawing of lines between spikes with small ISIs by using either Show Short ISI Lines in the context menu or by using the Tools Options menu command and selecting the Refractory Period tab You can set the Refractory ISI either in the Tools Options Refractory Period tab or in the Control Grid using the Refr ISI edit box 3 7 2 5 Cluster Positioning You can expand or contract the cluster image in feature space along the Xx R f R and y A A axes or move it left F right up or down by using the corresponding toolbar buttons or by using selections from the context menu Also you can right click and move the mouse up and down to Version 4 0 57 3 Views and Displays zoom the display in and out The display can be panned by left clicking holding the SHIFT key down and moving the mouse The zoom reset function amp in the toolbar or Reset Range and Zoom in the context menu resets the zoom and pan to the original range which is calculated so as to include all the points 3 7 2 6 Per View Toolbars Hints and Statistics Display The buttons displayed on the top and the operational hints and statistics shown at the top an
47. Software assigns all waveforms inside the arbitrary shape to that cluster When the Contours method is applied from the 3D view it operates in a 2D projection of the 3D feature space that is a standard perspective projection based on the chosen viewpoint That is the 3D feature space is first transformed to a 2D feature space screen space by using a standard perspective projection then the Contour sorting algorithm runs in screen space The other sorting algorithms can also be run in either 2D or 3D feature space Version 4 0 11 2 System Overview The Boxes sorting method requires placing two boxes on the Waveforms view to define each unit Each box thus specifies a region in time voltage space Any waveforms that intersect with both boxes for a unit are sorted into that unit The boxes can be resized and moved to any position on the Waveforms view The Lines sorting method is similar to the Waveform Crossing method except a unit can be defined as several lines segments that the waveforms must intersect The Bands sorting method is similar to the Template method see Semi automatic sorting methods below except that instead of using a single tolerance value for the entire unit the tolerance can be tailored to be different for different portions of the waveform This use of variable tolerances effectively defines a variable width corridor through which waveforms for a particular unit must pass You define the initial
48. Software can display any action potential waveforms spikes in a variety of displays including displays that show how the waveforms changed as a function of time during the experiment The spike data can be shown alongside simultaneously collected continuous and event data Extract and sort action potential waveforms spikes from continuously recorded data such as the data generated by Plexon systems including the Multichannel Acquisition Processor MAP Data Acquisition System PLX files and the OmniPlex D Neural Data Acquisition System PLX or PL2 files or by other data acquisition systems such as e Panasonic MED e Data Translation Offline Sorter Software detects spike waveforms within a continuous file using a threshold crossing method Thresholding for waveform detection can be applied to the e Raw voltage signal amplitude e Energy of the signal e Non linear energy of the signal Re sort action potential waveforms from previously sorted spike waveform data generated by Plexon systems such as MAP PLX files OmniPlex D System PLX or PL2 files or some other data acquisition system from the following list e Axion Biosystems e CyberKinetics e CED Spike 2 DataWave Technologies e Multi Channel Systems e Neuralynx The result of the sorting process is a set of timestamps that correspond to the spike times of the sorted units Offline Sorter Software can save these spike trains in the form of a NeuroE
49. Sources view provides a summary of the Sources available in the currently loaded data file A Source is a grouping of similar channels where all the channels in a Source have similar characteristics Some of these important characteristics are shown in the Sources view The columns shown in the Sources view are Name Every Source has a name For PL2 files acquired with the Plexon OmniPlex system the Source names should match those from OmniPlex Type A Source contains either Spike Continuous or Event data Chs The range of Offline Sorter channel numbers for the channels in this Source For spike and continuous Sources these channel numbers correspond to the channel numbers listed in the Channels view see Section 3 2 2 Channels View on page 29 Trodal For spike and continuous Sources this shows 1 for single electrode data 2 for stereotrode and 4 for tetrode data Freq The sampling frequency for the Source only relevant for spike and continuous Sources Id Every Source has a unique Id number These can be set up in Omniplex Raw Chs Every Channel has a unique identifying number within a data file this is called the raw channel number It generally does not correspond directly to the concept of a channel number within Offline Sorter Software Version 4 0 27 3 Views and Displays For some file formats the raw channel number does not need to be unique across all channels it only needs to b
50. Spike Sorting Step by Step The invalid waveforms disappear from the display To recalculate the principal components using only the valid unit clus ter data Select the Tools Recalculate the Principal Components menu command and rerun the principal component analysis using All valid waveforms Recalculate Principal Components Som Recalculate Principal Components Using Use Enhanced PCA Waveforms To Use All the waveforms All valid unsorted waveforms Use only waveforms within the time range Maximum waveforms to use 10000 Waveform Points To Use All the points Only the points between sort start and sort end Do not show this dialog again Gta 134 Offline Sorter Offline Spike Sorting Software Preparation For Sorting The resulting 2D Clusters view can resemble the following one J Info pA Cluster Display The three unit clusters white dots are now slightly better separated from one another than they were when the noise cluster was included in the PCA calculations Note You can undo the invalidation operation by selecting the Waveforms Mark All Waveforms As Valid menu command 4 4 2 3 Invalidating All Except The preceding invalidation procedures designated the waveforms to be invalidated You can invert that process to designate the waveforms to be retained by using the Waveforms Invalidate All Except Selected Waveforms setting When this
51. To save the waveforms and re sorted timestamps Select File Save As menu command Note You cannot overwrite the original file with this operation 4 8 1 1 Overwrite Backup Control on File Saves When files are saved using the File Save menu option or the toolbar Save button Offline Sorter Software provides control over saving files in terms of whether the data files are overwritten or backups are kept You can control this behavior using the File Save Behavior drop down list in the General tab of the Tools Options dialog box The options available are described in detail in Section 5 1 5 File Save on page 229 Version 4 0 205 4 Spike Sorting Step by Step 4 8 1 2 Exporting to a New PLX File Offline Sorter Software supports a flexible means to save timestamps and waveforms into a PLX file For information about this export command see Section 5 1 7 File Export to New PLX on page 231 4 8 1 3 Exporting to a New PL2 File Offline Sorter Software supports a flexible means to save timestamps and waveforms into a PL2 file For information about this export command see Section 5 1 8 File Export to New PL2 on page 232 4 8 2 Exporting to a NEX File The main result of the sorting process is a set of spike trains arrays of timestamps corresponding to the sorted units For information on analyzing these spike trains with NeuroExplorer see Section 5 1 9 File Export to NEX o
52. Used Tor S0riNg arrinin a 299 ooa Tomnpate PURI sraao aen EAS EE Ea 300 6 5 4 Adaptive Templates Algorithm siiiscscticadsscccnceesaiacstrsendiaddheoederaasetennsidddnropsaiausates 302 B00 R Means Agor eeii donteebecnieitiarenavened 303 650 Sanda d EMANGO E 303 6 5 7 Valley Seeking Algorithm ossirnonan inoino eaaa iiaii iia 304 6 5 8 T Distribution E M Al GRIM ssxiccscassrrdecendannrtantccnsnesciaasnniesanansntaciacssnerekuainies 305 Version 4 0 275 6 Reference Topics PE ETE ES EOE E N EA A A aaa iN 306 opnomd Gual ES siie a SA Sa AA 306 6 6 1 MANOVA F and eve noniine iirinn aeaea aE 307 Ea aN E E A T A E E T E A E E eee 308 Hoe Fe e E a T EE A NA E AA EE E E E 308 fe we Davies MONI onerarias 309 0S DUM e E E E E E A T T S 309 6 6 6 Per Unit Sort Quality Mei ssnsismraniinsnnaunninn 309 6 7 Thresholding Algorithm Aligned Extraction ccccsscccseeeseecceeeeeeeceeeeeeeeeceeeeenees 310 6 8 Enhanced PCA Feature Space coisa ssusidadicesiaiaceoictuiadegiceadeiegsie a E in aaae 312 6 9 Batch FIO PROCS SSG cicir 313 69 Bachi File Commn Sania cer ee epee reer rrp ner recnrrem tre reer rerrreT ey 313 6 0 2 Example Batch Command File sossen eiee 321 OAS NOS ia OE 322 6 10 Sample Batch Command Piles urnur a annedaa eins 324 PATI SUAN We O ciiai a a Aaa 324 6 10 2 RemArtiiecisSonAnGeEx Gort On sv eccisccccasorcdestaoydissebvesiacesnesanniegeesaaetucasorasialecnas 325 GUS eters Vale it eo cae 8 eee ee meee a a aai 325 6 10 4
53. Z axes from the drop down lists on the toolbar Ix Axis PC 1 v Axis PC 2 z Axis PC 3 Note Only the active features appear in the drop down lists which are a subset of all features Use the Active Features tab of the Tools Options dialog box to select which features are active For more information see Section 3 7 2 Clusters Views Common Functionality on page 54 When switching channels within the currently opened data file the selected features can either be remembered on a per channel basis or the same feature space can be used across all channels You can control this by the Remember selected features for each channel checkbox in the Tools Options Feature Calc dialog box If the sorting algorithms are used remember that they work differently in 2D and 3D feature space The feature space that the algorithms use depends on the active view when the sorting operation initiates See Section 6 5 1 Feature Space Used for Sorting on page 299 4 5 Spike Sorting Finally the waveforms can now be sorted into units Offline Sorter Software offers several methods to accomplish this 4 5 1 Manual Semi Automatic and Automatic Clustering Methods Offline Sorter Software offers manual semi automatic and automatic methods to identifying units or clusters Version 4 0 143 4 Spike Sorting Step by Step Manual Clustering Methods With these methods the user manually specifies the unit assignm
54. a Unit 1 Select the unit by clicking it s template in the Units view 152 Offline Sorter Offline Spike Sorting Software Spike Sorting 2 Click the Delete Unit button in the toolbar or select the Units Delete Selected Unit menu command or right click in the Clusters view and select the Delete Selected Unit menu command Use ALT right click in the 3D Clusters view The selected unit is deleted Note Other units can shift down after the unit is deleted For example if Unit a and Unit b existed prior to deletion when Unit a is deleted Unit b becomes Unit a To delete all the sorted units Select the Units Delete All Units menu command or hold down the ALT key and right click in the Clusters view and select the Delete All Units menu command 4 5 3 Manual Clustering using the Waveform Crossing Method The Waveform Crossing method of sorting is always available no matter which Sort Method is selected in the Control Grid This is different from pre 3 0 versions of Offline Sorter Software Pressing the Add Unit button on the toolbar of the Waveforms view will initiate Waveform Crossing sorting Holding down the Control key while clicking and drawing in the Waveforms view is also a shortcut to creating a new unit using the Waveform Crossing method 4 5 3 1 Adding a Unit This procedure identifies a subset of waveforms that comprise a unit To add a unit 1 Click Add Unit amp Fe k mov
55. an E mail to support plexon com containing the contents of the log Please only send this E mail if directed to do so by Plexon support personnel Note that there is an additional level of detail for log messages that by default do not show up in the message log Select Show Debug Messages from the right click menu to cause these more detailed messages to appear in the message log Version 4 0 37 3 Views and Displays 3 3 Control Grid ControlGrid 40X Channel sig01 Data Type Single Sort Method Valley Seek Zoom 1 0 Max wifdraw 100000 Sort Start 0 Sort End 1000 Slice 1 us 0 Slice 2 us 0 Slice 3 us 0 Slice 4 us 0 Outlier Thr 1 30 Parzen Mutt 1 00 Waveform View All Cluster Views As above Timeline View As above Only Segment Range from s 0 000 _ Range to s 74 892 Draw Grid 7 Draw Thr E 7 RefriSi us 1000 ISlHistMax ms 100 invalid wif Show Electrode Enable N A Use the Control Grid to set the following parameters Channel is the currently active channel Click to show a drop list of all the available channels The channel can be selected by double clicking it in the Info Grid Views For more information see Section 3 2 Info Grid Views on page 25 Data Type is the electrode recording mode Single indicates that each channel is viewed separately Stereotrode indicates that channels are arranged in groups of two Tetrode indicates that channels are arranged
56. beginning of the file For each spike waveform w i that is encountered in the file and that is sorted into the unit under consideration because it is within the Fit Tolerance of the current template t i the adaptive template algorithm will add that spike waveform to the average waveform to get an updated average s i s i 1 W w i W using a weighting factor W that is the adaptive template Weight mentioned above Then the average waveform s i is compared with the current template waveform t i by calculating the sum squared difference between s i and t i N _ ps1 d SO 1 where N is the number of samples in the waveform 302 Offline Sorter Offline Spike Sorting Software Details of the Sorting Algorithms If the calculated quantity d is greater than the adaptive template threshold value then a new template will be put into effect The new template waveform t i is set equal to the current average waveform s i The timestamp of the waveform being processed when this occurs is recorded as is the new t i so that Offline Sorter Software can display a history of the template waveform changes made during Adaptive Template Sorting Note that for small values of the adaptive template Weight W the average waveform changes only very slowly when the waveforms trend away from the template whereas for values of W near 1 the average waveform responds rapidly to shifting waveforms Also a small value of the adapti
57. better visualize the data and to find the viewing angle where cluster separation is maximized For the 3D Clusters view the Z axis is initially into the screen All the points are contained inside a box with grid lines to provide a frame of reference The dashed grey lines represent the X Y and Z axis and they intersect at 0 0 0 in feature space The solid grey lines show the projection of the X Y and Z axes onto the reference box The axes projections onto the box are labeled with the feature name represented by that axis Components in the 3D display such as the box wall the box top and bottom and the grid lines on the box can be shown or hidden using the Display tab under the Tools Options dialog Their colors can be set using Colors tab The grid lines provide only cues they do not correspond to actual values of features The number of grid lines drawn can be controlled in the Tools Options Display tab Version 4 0 65 3 Views and Displays Positioning the Eyepoint The eyepoint can be positioned with the following procedure To rotate the eyepoint Click and hold the left mouse button The pointer changes to a hand Mouse movement in the left right direction then rotates the eyepoint around the y axis and movement in the up down direction rotates it about the x axis pitch the view up down To move the eyepoint nearer or farther away Click and hold the right mouse button The pointer changes t
58. by sub keywords or arguments A will comment out a line and lines are not case sensitive Blank lines are ignored There are only six main keywords File Dir ForEachFile ForEachChannel Set and Process But the ForeEachFile ForEachChannel and Set commands have may sub commands In general an Offline Sorter batch file does the following things e pre specify the files to process using the File or Dir commands Version 4 0 313 6 Reference Topics e pre specify the actions to be done on each channel of each file e g do some sorting and the actions to be done on a file by file basis e g save the results using the ForEachChannel and ForEachFile commands e pre specify the values of other parameters that will affect how Offline Sorter Software performs the tasks e g set the Parzen Multiplier for Valley Seeking Sorting using the Set command e start all the processing with the Process command When running Offline Sorter Software the Help Quick Batch Reference menu item will display a convenient printable summary of the batch commands and their arguments The batch command keywords are 6 9 1 1 File The File command queues a file for processing The file is not actually processed until the Process command is encountered Specify the full path to the files Syntax File filename e g File c myfiles test p x will queue the file test plx for processing 6 9 1 2 Dir The Dir command queues all of t
59. can display the p value along with all the other sorting quality statistics in the Clusters views by selecting Show Statistics in the right click menus in the Clusters views Sort Quality Metrics The next section of the window lists the values of the Sorting Quality Statistics which are detailed on page 306 for both the 2D and 3D feature spaces Per Unit Sort Quality Section The next section is a table that shows the values of the L Ratio and Isolation Distance sort quality parameter for each unit for both the 2D and 3D feature spaces Per Unit Statistics Section The next section is a table that presents the means and standard deviations of the multivariate normal distributions for the waveforms of each unit for both the 2D and 3D feature spaces The percentage of waveforms in each unit that do not meet the minimum ISI interspike interval period is also displayed Normality Statistics Section The next section is a table that calculates the Skew and Kurtosis characteristics of the multivariate normal distribution for groups of waveforms all valid waveforms the unsorted waveforms and the waveforms for each Unit for both the 2D and 3D feature spaces Large values of Skew and or Kurtosis indicate that the data is not well fit by a multivariate normal distribution Some analyses have underlying assumptions of normally distributed data e g the Multivariate ANOVA analysis Pairwise Sort Quality Section This section contains a num
60. can write code to make use of the Neuroshare DLL to in principle read any file format for which the vendor has supplied a Neuroshare DLL The import of some formats is dependent on the presence of a Neuroshare DLL that is separately downloaded and installed on the computer In general Plexon cannot fix problems in the Neuroshare DLLs supplied by other vendors During Offline Sorter testing we observed that unfortunately there is much variation between the Neuroshare DLLs produced by different vendors The Neuroshare loader in Offline Sorter tries to be as general and tolerant as possible but in most cases the loading of the data has problems e g the gains or reported voltage ranges are not correct or the pre threshold time is not available Note Vendors usually supply both 32 bit and 64 bit versions of their Neuroshare DLLs The appropriate 32 vs 64 bit version of the Neuroshare DLL must be used with the 32 or 64 bit version of Offline Sorter Note Files loaded using a Neuroshare DLL cannot be saved via the Save or Save As functionality You must save these files using one of the Export functions These files also do not react to the Remove empty spike channels setting which is in the Tools Data Import Options dialog box Version 4 0 221 5 Menu Options The following sections provide information about each of the file formats that Offline Sorter Software can read 5 1 2 1 Alpha Omega Import for Alpha Omega
61. channels Show MANOVA Statistics V Show Cluster Quality Metrics 33 W Dunn V PseudoF V L Ratio V Davies Bouldin Isolation Distance V Show Per Unit Statistics Vv L Ratio V Isolation Distance V Show Unit Templates V Show 2D Cluster View en C Individual sorting quality statistics can be suppressed if they are not of interest as can entire sections of the report Version 4 0 87 3 Views and Displays 3 14 2 Output to PowerPoint The Sorting Summary view can be sent to PowerPoint by selecting Send To PowerPoint in the right click menu in the Sorting Summary view For this feature to be available PowerPoint must be installed on the computer Sorting Summary for 071005a plx Channel 10 File Name CPlemnDataeO71005e pix Unita Time amp Dat 100509 14 02 40 from header Waveform Length 0 usec Prethirshold 200 wee AD Freg 400000 Hz Number of Unite 3 Sred 1219220422 Using K Meae Features 2D PC 1 PC 2 3D PC 1 PC 2 PC3 Multivariat ANOVA Incbading Uraorted Waveforms as a Grop In 2D Cluster Space FGA9SM 12568945 P 365 04 In JD Cluster Space FO 60262 10622635 P 1242 eO16 Couns All Vaid WFe 31500 Unsored Wis 1266 Unita 11165 Unit b 15628 Unite Chetr Validity 2 J Pseude F Davies Bouldin 07200 Dwa When the Send To PowerPoint menu item is selected PowerPoint opens if it is not already open and a ne
62. channels without warning until the set ting is unchecked 326 Offline Sorter Offline Spike Sorting Software Large File Tips When saving a NeuroExplorer NEX file by default Offline Sorter Software must read the continuous data channels in order to write them to the NEX file which can be time consuming If the continuous data channels are not desired to be present in the NEX file you can disable writing the continuous data channels by unchecking the Export continuous data channels checkbox in the Export to NEX Options dialog box 6 11 5 Initial PCA Calculation By default Offline Sorter Software performs a PCA calculation on each channel after it is loaded into memory However this calculation is wasted if the sorting is to be done in a feature space that does not involve principal component projections e g using peak heights to sort tetrode data You can effectively turn off the PCA calculation by entering a value of 0 in the edit box under Maximum Number of Waveforms to Use in the PCA Calculation in the Feature Calc tab of the Options window Version 4 0 327 6 Reference Topics 328 Offline Sorter Offline Spike Sorting Software Plexon Inc Appendix A Navigating the Plexon User Interface Fel Plexon User bite iiair A 2 A2 Sereen EIS SIG esna A 3 AT ETU Sn A 3 Aee TONO oa A 5 Paa INOW S aaa aaa aea a aise A 6 A 3 Standard Menu Items and DialogS ccscceeeeeee
63. detection See Waveform Detection View on page 74 Non Linear Energy Sri v t v t 1 v t 1 ISI Previous ISI Next For waveform i ISI Previous is the amount of time in mSecs between the waveform i 1 and i while ISI Next is the amount of time between waveform i and i 1 Both values are capped at 9999 mSecs to avoid large gaps from over extending the range of values Timestamp The timestamp of the waveform in the file Note Selecting the timestamp as one of the dimensions in the Clusters views allows the viewing of the other feature s as a function of time Discontinuities or drifts in time can indicate a change in the electrode position or the recording conditions Per Electrode Features Only available for stereotrode or tetrode data Most of the above features are available calculated per electrode For example the feature Peak EL 1 is the peak value of the waveform within the first electrode in a stereotrode or tetrode Peak EL 2 is the peak value within the second electrode of the stereotrode or tetrode For further discussion see Section 6 3 Stereotrode and Tetrode Data on page 291 Per Electrode Ratio Features only available for Stereotrode or Tetrode data These features are formed by taking the ratio of Per Electrode feature values For example the feature Peak EL 1 EL 2 is Peak EL 1 divided by Peak EL 2 Ratios with a denominator value of 0 are arbitrarily s
64. digital event data e There is an assumption that raw channel numbers within a Source are contiguous without gaps When loading a Plexon PL2 file the Sources that appear in Offline Sorter Software will be the Sources that were directly written to the PL2 data file However when loading a data file from any other supported data file format Offline Sorter Software will still view the contents of the data file as if it Version 4 0 277 6 Reference Topics contained Sources It will create Sources based on the contents of the file For example suppose Offline Sorter Software loads a non PL2 file that contains 64 spike channels 64 continous channels recorded at 20Khz and 8 continuous channels recorded at 1Khz Offline Sorter Software will create 3 Sources a spike Source containing 64 channels a continuous Source with 64 channels that has a digitization frequency of 20Khz and another continuous Source with 8 channels that has a digitization frequency of 1Khz 6 1 2 Offline Sorter Channels Offline Sorter Channels referred to simply as channels elsewhere in this manual are shown as rows in the Channels view See Section 3 2 2 Channels View on page 29 An Offline Sorter Channel is uniquely identified solely by its channel number Offline Sorter Channel numbers always start at 1 and are contiguous without gaps Only one Offline Sorter Channel can be the current channel loaded into Offline Sorter Software at a
65. e Customize Clicking Customize displays the Customize dialog box as shown below Customize Toolbars Commands Keyboard Run Menu Options Toolbars Standard VJEdit _ Animate Looping Find Fool V Views For details on using the Customize dialog box see Section A 4 Customization on page 18 Offline Sorter Offline Spike Sorting Software A 3 2 Run Menu The purpose of the Run menu is to launch other applications quickly The Run menu will be populated by default with some common Plexon applications If you wish to be able to launch other applications from within the current application you can add new entries to the Run menu A sample Run menu is shown below For additional information about customizing the Run menu see Section A 4 4 Run Menu Customization on page 21 NeuroExplorer 2 l Explorer A 3 3 Help Menu The Help menu contains four items Help Quick Reference Web Update and About XXXXX where XXXXX is the name of the application Help Help Quick Reference Web Update About CinePlex Markup Help Clicking Help displays the User s Guide for the application e Quick Reference Quick Reference may be missing in some applications When present clicking Quick Reference displays a dialog box similar to the one shown below The purpose of the Quick Reference dialog box is to show keys and mouse buttons that control some of the co
66. either in the Waveforms view e See Adding Waveforms to Selected Units in the Waveforms View on page 154 and e Section 4 6 3 Cleaning the Selection on page 187 e or in the Clusters views e See Adding Waveforms to Selected Units in the Clusters Views on page 150 and e See Removing Waveforms from Selected Units In the Clusters Views on page 151 4 5 9 Semi Automatic Clustering using Standard E M Select the Standard E M method of sorting by selecting Std E M in the Sort Method drop list in the Control Grid or by selecting the Sort Change Sort Method Use Standard E M Sorting Method menu item The Standard E M algorithm assigns waveforms to a pre determined number of clusters units using an iterative algorithm operating on the waveform points in feature space For more details see Section 6 5 6 Standard E M Algorithm on page 303 The Standard E M algorithm operates differently depending on which view initiates it and which options are set see Section 6 5 1 Feature Space Used for Sorting on page 299 There are two steps involved 1 Selecting initial cluster centers This is done in exactly the same fashion as for the K Means algorithm described above See Section 4 5 8 1 Selecting Initial Cluster Centroids on page 171 for a step by step description 2 After the cluster centers have been specified start the algorithm To run the Standard E M clustering algorithm e Click Find Units in the toolb
67. etc For more information see Section 4 8 3 Exporting Information on page 206 2 4 2 4 Exporting Per Channel Data To accommodate later analyses of the data Offline Sorter Software can write out selected information about each channel into either a text or a MATLAB file or into an Excel spreadsheet For a given channel any of the sorting quality statistics can be exported and also the unit counts sorting criteria etc For more information see Section 4 8 3 Exporting Information on page 206 2 4 2 5 Exporting Per Unit Data To accommodate later analyses of the data Offline Sorter Software can write out selected sorting information about each sorted unit into either a text or a MATLAB file or into an Excel spreadsheet For a given unit it is possible to export items like cluster center positions cluster sigmas ISI percentages etc For more information see Section 4 8 3 Exporting Information on page 206 2 4 3 Reproducing Results Offline Sorter Software provides several methods to apply or re apply sorting criteria to data 2 4 3 1 Export Sort Info to TPL The sorting criterion used in sorting channels can be written to a TPL file The TPL file remembers information such as the features used for sorting the sorting method and any contours templates or parameters that control or affect the sorting For more information see Section 4 8 4 1 Saving Templates on page 212 2 4 3 2 Sort Using TPL file
68. export If a current channel is selected in Offline Sorter Software when selecting File Export Per Unit Data the default is Current Channel only which means Offline Sorter Software exports all units Version 4 0 211 4 Spike Sorting Step by Step on the current channel You can also export all units on all channels to a single file Select What to Export for Each Channel This enables selection of what items appear in each column for each exported row of data The Available list box on the left shows all the items that can be exported and the Chosen list box on the right shows those that have been selected for export Click on one or more items to highlight them and move items back and forth between the list boxes by then pressing the Add gt gt or lt lt Remove buttons Use other convenience buttons to add all items remove all items or reset the chosen items to the factory defaults The order that the items appear in the Chosen list box controls the order that they appear in the output file You can change the order by selecting one or more items in the Chosen list box and pressing the Move Up or Move Down butions Offline Sorter Software remembers the chosen items and their order so that the next time information is exported the same items are chosen automatically The items available include the Channel number unit number number of waveforms the values of the normality statistics mean sigma skew and kurtosis for
69. file from another vendor s hardware Select File Import menu command and select the vendor 110 Offline Sorter Offline Spike Sorting Software Waveform Acquisition From Continuously Recorded Data To open an arbitrary binary file with continuously digitized data header plus multiplexed A D values stored as 2 byte signed integers 1 Select File Import Binary file with Continuously Digitized Data The Import Binary Digitized Data File dialog box opens Import Binary Digitized Data File File To Import Import Parameters Number Of Channels Data Offset from the start of the file bytes Digitizing Frequency H2 4 D Converter Resolution bits 12 Maximum Yoltage m 10 Swap Bytes Load from ofi File Save to ofi File Load Using Companion ofi File Cancel 2 Enter a file name and the following information number of channels data offset digitization frequency A D conversion resolution and maximum voltage To perform byte swapping on the file click Swap Bytes 3 Offline Sorter Software can save the import settings to an ofi file which can be recalled later using the Save to ofi File and Load from ofi File buttons If an ofi file with the same base name of the File To Import exists it can be quickly loaded by pressing the Load Using Companion ofi File button 4 2 2 Selecting the Continuous Data Channel Offline Sorter Software can optionally load
70. first column See Section 6 1 Details of Channels and Sources on page 6 277 Version 4 0 31 3 Views and Displays 3 2 4 File Summary View The File Summary view contains various items of interest for the currently loaded file in a two column spreadsheet format File Parameters e File Name shows the full path to the currently loaded file on disk Time amp Date from header shows the time and date that the data was collected as the data acquisition software wrote the information into the file 32 Offline Sorter Offline Spike Sorting Software Info Grid Views header If the time and date is unavailable from the file header the time amp date of the file itself appears Comment lists the comment field in the file header if present Note Some file formats notably PL2 make a distinction between a Creator Comment a comment entered when the file was created acquired and Reprocessor Comment a comment added when the data file was later processed File Size bytes indicates the size of the currently loaded file in bytes Experiment Length sec is the total amount of data in seconds that is in the current file Format Version shows for some file formats the version of the file format currently loaded Acquiring Software shows the software that was used to acquire the data This parameter only appears when a PLX or PL2 file is loaded Timestamp Frequ
71. first unit starting with Unit a for which it passes through the band If a waveform passes through the band for several units it is assigned the lowest lettered unit 4 5 8 Semi Automatic Clustering using K Means Select the K Means method of sorting by selecting K Means in the Sort Method drop list in the Control Grid or by selecting the Sort Change Sort Method Use K Means with Cluster Centroids menu item The K Means algorithm assigns waveforms to a pre determined number of clusters units using an iterative algorithm that operates on the waveform points in feature space For more details see Section 6 5 5 K Means Algorithm on page 303 The K Means algorithm operates differently depending on which view it is initiated from and some options settings see Section 6 5 1 Feature Space Used for Sorting on page 299 There are two steps involved 1 Section 4 5 8 1 Selecting Initial Cluster Centroids on page 171 2 Section 4 5 8 2 Running the K Means Clustering Algorithm on page 173 4 5 8 1 Selecting Initial Cluster Centroids For the K Means algorithm to work it is necessary to specify e how many clusters there are e the approximate cluster centroids Version 4 0 171 4 Spike Sorting Step by Step To define a cluster center 1 Move mouse pointer to the Clusters view 2 Point to the center of the cluster 3 Double click the left mouse button or hold down CTRL and single click Control left click
72. for export Click on one or more items to highlight them and move items back and forth between the list boxes by then pressing the Add gt gt or lt lt Remove buttons Use other convenience buttons to add all items remove all items or reset the chosen items to the factory defaults The order that the items appear in the Chosen list box controls the order that they appear in the output file You can change the order by selecting one or more items in the Chosen list box and pressing the Move Up or Move Down butions Offline Sorter Software remembers the chosen items and their order so that the next time information is exported the same items are chosen automatically The items available include the channel number the number of units waveforms and unsorted waveforms in the channel the sort method used to sort the channel and the values of any of the Sorting Quality Statistics both 2D and 3D You can also export the features selected for each axis of feature space as a numerical code Select What File to Store the Exported Data in by typing a filename or selecting one by using the Browse button To append the data onto the end of an existing file click Append if existing Click Export For exports to Excel save the spreadsheet using Microsoft Excel 210 Offline Sorter Offline Spike Sorting Software Saving the Sorting Results 4 8 3 3 Exporting Per Unit Data Selecting the File Export Per UnitData menu selecti
73. iaaa aaaea 198 AE al Wavelonm INS PE CHON soiecccaicnindies auledarceenledaomeniedntaatiadedeuiidesletads 198 4 7 2 Examining Sorting SIGE siisii aa 200 4 7 3 Examining the Sorting VS Time sessssssesseeseireesresssrrssrnerressressrens 204 4 8 Savmg the Sorting RESUS ameona 205 4 8 1 Saving Timestamps and WaveformS s ssssssesreesseeesseesreensreesrens 205 482 EXPO Mey TO a NEX PIS sennsssrnarnsn 206 4B EHNE Mormaii ea 206 484 Saving Template FileSend aeia ian 212 4 8 5 Saving and Restoring Scan Results sssisisissiiriiriiiieniissniiniinna 214 108 Offline Sorter Offline Spike Sorting Software Procedure Summary 4 1 Procedure Summary The spike sorting process with Offline Sorter Software involves the following general procedures Waveform Extraction can be omitted depending on type of data files Preparation for Sorting Spike Sorting Unit Selection Waveform Inspection and Unit Cleaning Saving Sorted Spike Times Offline Sorter Software is capable of sorting action potential waveforms spikes in either continuously digitized data or in previously sorted spike waveform data files re sorting For sorting spikes in Continuously Recorded data 1 Open the continuously digitized data file See Section 4 2 1 Opening the Digitized Data Files on page 110 2 For each of the channels that contain spike data Select the data channel See Section 4 2 2 Selecting the Continuous Data Channel
74. in microvolts ContPreampGain allows setting the preamp gain to be used for all continuous channels SpikePreampGain allows setting the preamp gain to be used for all spike channels DetectDead sets the number of samples to ignore following a detection the dead time to use for ForEachChannel Detect 318 Offline Sorter Offline Spike Sorting Software Batch File Processing AlignType determines how the waveforms will be aligned 0 first local max 1 first local min 2 first local extremum 3 global max 4 global min AlignMaxShift is the maximum amount a waveform will be shifted in clock ticks AlignDuringDetect toggles the align during detection feature on off ArtifactWidth the number of samples used to detect coincidence for ForEachFile InvalidateArtifacts ArtifactPercentage the percentage of channels that must have coincident spikes for ForEachFile InvalidateArtifacts BetweenEventl1 BetweenEvent2 event names to use for KeepWfsBetweenEvents interval filter BetweenEvent1 Offset BetweenEvent2Offset time offsets in msec to use for KeepWfsBetweenEvents interval filter Filter will invalidate waveforms outside of the time interval between time of BetweenEvent BetweenEvent Offset to time of BetweenEvent2 BetweenEvent2Offset SpanEvent event name to use for KeepWfsInSpan interval filter SpanEventStartOffset SpanEventEndOffset time offsets in msec to use fo
75. inter spike intervals ISD less than the Refractory Interspike Interval You can set the value for the Refractory ISI in the Control Grid or through the Refractory Period tab on the Tools Options dialog box You can use the Units view to inspect the interspike interval related aspects of the current units The top row of graphs of this display shows the template average waveform for the unit solid line as well as 3 standard deviations from the template dash lines You can use the edit controls with spin buttons to adjust the tolerances for Template Sorting Channeldda The bottom row of graphs of this display shows the interspike interval histograms The X axis of each histogram is from zero to ISIHistMax a value that may also be set in the Control Grid The total number of spikes for each unit is also shown 190 Offline Sorter Offline Spike Sorting Software Unit Cleaning The percentage of spikes with interspike intervals less than the Refractory Interspike Interval appears in red and a red bar indicator also appears in the upper portion of the Units view You can change the value of ISTHistMax and Refractory Interspike Interval in the Control Grid You can also set the Refractory Interspike Interval value by using the Tools Options Refractory Period tab Offline Sorter Software provides a means for visualizing which spikes in a cluster have inter spike intervals less than the refractory period and
76. invalidated This is a equivalent to pressing the InvalidateWfs_ button on the per view toolbars or the button on the main toolbar 5 5 8 Waveforms Invalidate All Except Selected Waveforms Selecting this menu item will put the Waveforms view and Cluster views into the mode where the user is expected to cross on the Waveforms view or circle on the Cluster views the waveforms that should NOT be invalidated This is equivalent to pressing the a button on the main toolbar 5 5 9 Waveforms Mark All Waveforms As Valid Selecting this menu item will change all waveforms that are currently marked as being invalid to again being valid and unsorted waveforms 5 5 10 Waveforms Invalidate All Waveforms in Selected Unit Selecting this menu item will invalidate all waveforms that have been sorted into the currently selected Unit This same function is also available from the Units menu 5 5 11 Waveforms Perform Digital Referencing on Continuous Data Select this menu option to digitally subtract a reference channel from all channels in a continuous Source This is useful to reject common mode noise from the signal The reference channel can be created by averaging together a collection of other channels using either the Common Average Reference CAR or Common Median Reference CMR techniques Selecting the Waveforms Perform Digital Referencing on Continuous Data menu item displays the Digital Referencing dialog
77. large Closer to zero the converse is true the extraction produces a large number of spikes but many of the spikes are not distinguishable from noise and so the SNR is very low This graph may be useful in helping the user decide on the trade off between a large number of spikes and a high SNR 3 22 Overlaps Analysis View In some recording situations where the neural firing rate is high spikes can be lost from the analysis because of overlapping waveforms That is one neural firing results in a threshold crossing and initiates the capture of a spike but before spike window is completed i e before all the samples within the Waveforms window have been collected another neural firing is picked up on the same electrode This results in an overlapping waveform and these overlapping waveforms will have a spike waveform that is not shaped like the unit template of either of the neurons involved These overlapping waveforms typically show up as outliers and usually are not sorted into any unit by automated sorting algorithms Version 4 0 99 3 Views and Displays The Overlaps Analysis view in Offline Sorter Software can be used to help rescue some of these overlapping waveforms by first detecting that a given waveform may have been produced by overlapping unit firings and then resolving them by reconstructing the non overlapped versions of the two overlapping spikes This then allows the formerly overlapping spikes to be p
78. left hand list box Select one or more features in either list box then press the Add gt gt or lt lt Remove buttons to transfer features between list boxes The order in which the active features show up in the drop down feature lists is the same as the order in the Currently Active list box Re order the items in this list box by selecting one or more features and pressing the Move Up or Move Down buttons Pressing the Reset to Defaults button reverts the set of active features to the as shipped defaults Some of the features are relevant only for stereotrode or tetrode data these features although they can be active do not show up in the drop down feature lists when single electrode data is being viewed in Offline Sorter Software Version 4 0 55 3 Views and Displays 3 7 2 2 native loaderWaveform Highlighting The Clusters views allow viewing the waveform shape corresponding to any point in the display To view the waveform 1 Left click in the 2D Clusters view 2 Left click while holding down the ALT key in the 3D Clusters view The pointer becomes crosshairs the nearest point in the Clusters view is highlighted with a box and the corresponding waveform is shown in the Waveforms view to the left see the red arrows in the diagram below If the waveform is in the time range being displayed the selected waveform is also highlighted with a white box in the Timeline view 2D Clusters Display 16CHS i l E3 If t
79. menu 237 electrodes disable or ignore 294 E M algorithm 303 energy calculations 77 enhanced PCA 297 312 Events view 36 Expectation Maximization algorithm 303 exporting information 206 exporting results 14 exporting timestamps 231 exporting to NEX file 233 exporting to PL2 file 232 exporting to PLX file 231 extracting waveforms 123 F Feature vs Feature view 80 features 8 feedback statistics 203 file import 9 221 File menu 220 file save options 229 File Summary view 32 frequency spectrum 80 G gain adjusting 115 H help Plexon technical support 5 Help files and quick key reference 273 Index 2 Help menu 273 Help Topics online user guide 273 ignore electrodes 294 importing data files 9 110 221 Info Grid Views 25 installation 2 interpolation methods 114 invalidating waveforms 129 ISI Histograms view 90 K K Means algorithm 303 L large files working with 325 license keys 3 license management 3 MAD median absolute deviation 301 MANOVA F and p values 307 median absolute deviation MAD 301 menu options 217 Message Log 37 Messages view 37 N NeuroExplorer 9 O outputs from spike sorting 14 overlapping waveforms 99 overlapping waveforms rescuing 194 Overlaps Analysis view 99 overview 8 Offline Sorter Offline Spike Sorting Software P partial sort 306 PCA Results view 88 PCA view 36 PCA enhanced 297 312 PCA principle components analysis 88 per unit
80. noise sigma values obtained from this procedure are not heavily dependent on the arbitrary choice of 2 7 full signal sigmas used as the signal rejection criterion Note that the noise sigma will always be less than the full signal sigma 3 11 2 Adjusting the Threshold for Waveform Detection The threshold can be changed by clicking and dragging the solid red line either on the Waveform Detection view or on the Timeline view Thresholding can be done on either positive or negative amplitudes Alternately a threshold can be set by entering a value in the Thr edit box in the Timeline view or by clicking on the associated spin control 3 11 3 Changing the Detector The waveform detection algorithm can use thresholds on quantities other than raw waveform amplitude As explained in Section 4 2 6 Specifying the Waveform Detection Options on page 120 the Detector and Waveform Options menu dialog can be used to select the signal threshold to be the e Raw Voltage Signal the default or e Energy of the Signal or e Nonlinear Energy of the Signal or e Signed Energy of the Signal When a signal is selected other than Raw Voltage amplitude for use in spike detection the Waveform Detection view and the Timeline view show both the selected parameter top display and the Raw Voltage bottom display Shown 76 Offline Sorter Offline Spike Sorting Software Waveform Detection View below are the Waveform Detection view an
81. on page 111 Specify up sampling if needed See Section 4 2 3 Specifying Up Sampling on page 114 Check and adjust gains if needed See Section 4 2 4 Adjusting Gains on page 115 Specify the low cut digital filter if needed See Section 4 2 5 Selecting a Digital Filter Low cut or High cut on page 118 Specify the waveform detection options See Section 4 2 6 Specifying the Waveform Detection Options on page 120 Set the threshold and extract the waveforms See Section 4 2 7 Extracting the Thresholded Waveforms on page 123 Go to Step 3 on page 109 For Re sorting Spikes from Previously Sorted Waveform spike Data files 1 Open the spike waveform data file See Section 4 3 1 Opening the Spike Waveform Data File on page 124 Go to Step 3 on page 109 2 Convert Data Type if necessary See Section 6 3 Stereotrode and Tetrode Data on page 291 3 For each channel with spike waveforms Inspect the data See Section 4 4 1 Data Inspection on page 127 Remove artifact waveforms See Section 4 4 2 1 Marking Artifact Waveforms as Invalid using Waveforms View on page 131 Version 4 0 109 4 Spike Sorting Step by Step If necessary select intervals of valid data See Section 5 8 6 Tools Interval Selection on page 264 Check Waveform Alignment if necessary See Section 4 4 4 Waveform Alignment on page 138 Adjust Sort Width if necessary See Section 4 4 5 Adjus
82. printable so that it can serve as a permanent record of the sorting The contents of the Sorting Summary view can also be exported to PowerPoint Much of the information on the Sorting Summary view can be found in other views or windows in Offline Sorter Software the value of the Sorting Summary view is that it gathers the most important sorting information together in one view To display the Sorting Summary view Select the View Sorting Summary menu command or press the ES button on the toolbar 84 Offline Sorter Offline Spike Sorting Software Sorting Summary View Sorting Summary for Bkr34 plx Channel AD01 File Name C Workshop2013 Data Bkr34 pkk Time amp Date 02 18 02 11 09 30 from file Wif Length 600ysec Prethreshold 150 usec A D Freq 40000 0 Hz Number of Units 4 Sorted 07 06 14 14 08 06 Using Valley Seeking Features PC 1 PC 2 PC 3 2D 3D J3 6 91893 3 98766 Pseudo F 122128 70387 5 Davies Bouldin 0 298969 0 423413 Dunn 1 4469 0 88964 L Ratio 0 062544 0 068483 Iso Dist 13 5261 42 0353 Counts RMSE ISI lt 2000 pS L Ratio Iso Dist All Valid WFs 54079 97 1807 2D 3D 2D 3D Unsorted WFs 1121 184 885 Unita 19090 49 7084 1 493 0 008661 0 010114 62 1284 71 3751 Unitb 12617 51 0046 0 8322 0 052412 0 057963 32 12 70 1322 Unite 16536 52 5969 5 152 0 062544 0 068488 52 438 67 476 Unit d 87 7151 5 493 0 008617 0 020005 13 5261 42 0353 Unit b Unitc Unit d Range 6 mV
83. quality metrics versus the scanning parameter the Parzen Multiplier in the above screenshot The values of just one or of all sort quality metrics can be plotted on the Scan Graph view This is controlled by selecting Customize Display from the right click menu F Graph Customization What To Graph Color Symbol 7 Units E v arde 7 33 N v Grde v v Pseudo col Cirle v Davies Bouldin __ v Gide v Dunn Er Grde L Ratio ka Girde Isolation Dist Ew Cirde gt P Value M Grde Version 4 0 95 3 Views and Displays Checking the checkbox next to a sort quality metric will display that metric as a series of symbols connected by a line on the graph The color of the symbols and line can be selected as can the shape of the symbol Pressing the Reset to Defaults button will reset all values to the factory default values Offline Sorter Software has the concept of a sorting which is the unit assignment for every waveform in the channel At each step of the parameter scan Offline Sorter Software remembers the resultant sorting Left clicking in the Scan Graph view or on a result row in the Scan view will select the corresponding sorting and make it current and all other views will change to show that sorting The last selected sorting is shown by drawing a white rectangle in the Scan Graph view and by showing a gt gt in the
84. r LEXON Neurotechnology Research Systems Offline Sorter Offline Spike Sorting Software Version 4 0 April 2015 User Guide Plexon Inc 6500 Greenville Avenue Suite 700 Dallas Texas 75206 TU HARRI BRA S USA Ar ecaro ooy a T 02 2911 5233 F 02 2911 6855 E info kyst com tw A Caution Electrostatic Discharge Some devices can be damaged by improper handling Use appropriate electrostatic discharge ESD procedures when handling these devices See http www esda org for additional information on ESD procedures Offline Sorter Offline Spike Sorting Software User Guide Document Number OFSMNO0004a Document Status Standard Software Version 4 0 Date April 2015 Copyright 1983 2015 Plexon Inc All rights reserved Plexon Inc Proprietary The information contained herein is the property of Plexon Inc and it is proprietary and restricted solely to assist Plexon Inc customers Neither this document nor the contents may be disclosed copied revealed or used in whole or in part for any other purpose without the prior written permission of Plexon Inc This document must be returned upon request of Plexon Inc Information is subject to change without notice Plexon Inc reserves the right to make changes in equipment design or components as progress in engineering or manufacturing may warrant PLEXON Plexon the five line symbol CereStage CineCorder CineLAB CineLyzer Cin
85. represents the current channel mapping then edit the channel mapping in the ofm using a text editor to create the desired channel mapping This ensures for example that the ofm file will have the correct overall structure including the correct Source ID for the desired final mapping An ofm file can be saved that contains either just the currently selected Source or for all Sources To save an ofm file select Save Current Source Channel Map to a File or Save All Sources Channel Map to a File from the right click menu in the Channel Map view 6 3 Stereotrode and Tetrode Data 6 3 1 Loading Stereotrode or Tetrode Spikes Offline Sorter Software will automatically load stereotrode and tetrode spikes from most file formats that support stereotrode or tetrode data For most file formats including Plexon plx and pl2 what was 2 or 4 channels in the raw data file are combined together so that they appear to become a single stereotrode or tetrode channel in Offline Sorter Software When loading stereotrode or tetrode data from Plexon plx files Offline Sorter Software tries to match waveforms that appear on the individual channels that make up a full stereotrode or tetrode channel by timestamp That is the complete stereotrode or tetrode waveform consists of waveforms from the 2 or 4 channels that comprise the stereotrode or tetrode and those waveforms must have the same timestamp The Invalidate Incomplete Stereotrode and Te
86. row header in the Scan view In effect the selected sorting becomes the current sorting for the channel Left clicking in the Scan Graph view is a convenient way to compare quickly different sortings and select the most pleasing one as the final sorting of the channel The automatic sorting methods that utilize scanning see Section 4 5 13 Automatic Clustering Using the Scanning Methods on page 182 are in reality only doing parameter scans and so will fill the Scan view and the Scan Graph view as they run Offline Sorter Software can also save and restore the results of a scan to and from files see Section 4 8 5 Saving and Restoring Scan Results on page 214 To save or restore a set of scan results select Save Scan Results to a File or Restore Scan Results from a File from the right click menu of either the Scan view or the Scan Graph view A new scan can be set up and initiated by selecting Scan Parameters from the right click menu on either the Scan Graph view or the Scan view P Scan Type Seeks hime rau v Start 1 End 10 Step 1 Fixed Value D O F Mult 10 v Use 3D Feature Space V Normalize Sort Quality Metrics Start Scan Now C ok Cancel This dialog is equivalent to the top portion of the Scan view it sets the same values 96 Offline Sorter Offline Spike Sorting Software Sort Quality vs Time Segment Graph View 3 20 Sort Quality vs Time Segment Graph Vi
87. segments and digital event data Continuous includes both continuous signals sampled at high frequencies spike and wide band signals EMG etc and continuous signals sampled at low frequencies field potentials and other external signals such as x y eye position etc 2 5 3 Digital The term digital refers to digital events that Offline Sorter Software reads from the file Digital events in general can also have a strobe value for each event in addition to a timestamp These are often called Events as in the Events view 2 5 4 Fast and Slow The terms fast and slow describe sampling rates when there is a need to distinguish between two rates 2 5 5 Channel A channel is data recorded from an electrode or a set of electrodes for stereotrode tetrode Channels can contain either continuous or spike data or both The term can also sometimes be applied to digital events a digital event channel is the collection of all digital events with the same identifier or name Most views in OFS show data only from the currently selected channel 2 5 6 Source Channels are organized into Sources The concept of a Source should be familiar to users of Plexon s OmniPlex System it is a block of contiguously numbered Channels that all have certain shared characteristics For example all spike data channels in the same Source must have the same sampling rates and number of samples per spike Offline Sorter Offline
88. selected unit Additionally you can display the Time axis or the Counts axis or both using a log scale 3 17 Cross Correlograms View The Cross Correlograms view displays a half matrix of cross correlograms between all units and optionally the unsorted waveforms as well in a channel The firing of each unit is used as a reference time time zero to form a correlogram of a different unit s firings using an adjustable timescale The cross correlograms are arranged on a grid where the reference unit increases along the vertical axis and the plotted unit increases along the horizontal axis with the redundant mirror image correlograms omitted Version 4 0 91 3 Views and Displays To display the Cross Correlograms View Select the View Cross Correlograms menu command or press the Fa button on the toolbar bd i li EA Info a 2D Clusters ade Correlograms The parameters for the Cross Correlograms are set by using the right click context menu The view can be configured to show the cross correlograms formed by permuting either all sorted units all sorted units plus the unsorted waveforms or just the currently selected units The labeling can be enabled or disabled which is useful if there are large numbers of units Also whether or not the reference unit firing is counted in the correlogram can be selected The timescale and bin widths used to build the correlograms can be set using the Binning section of
89. setting is selected all waveforms that are not selected are invalidated This selection applies in both the Waveforms view cross the waveforms to be retained and in the Clusters views circle the waveforms that are to be retained 4 4 2 4 Automatically Invalidating Artifacts Offline Sorter Software includes a tool that automatically finds and invalidates artifacts that occur simultaneously or nearly so across several channels The Invalidate Artifacts tool only uses spike times The tool does not consider waveform shapes or amplitudes To use the Invalidate Artifacts tool two criteria must be set 1 atime width that ranges downstream from the first artifact threshold crossing point on any channel 2 apresence criterion that sets the number of channels on which a spike must be simultaneously present within the time range to be identified as an artifact When the tool determines that an artifact is present at time T on a sufficient number of channels the tool invalidates all waveforms that are present on all channels between the times T nin and Tmax where Tmin and T nax are the Version 4 0 135 4 Spike Sorting Step by Step minimum and maximum time stamps of the spikes in the artifact The tool runs on all channels not just the currently loaded channel To be identified as artifacts simultaneous spikes must be present on at least a critical number of channels Set the presence criterion in percent multiplied by the n
90. the MCStream DLL COM object being installed and properly registered on the system If it is not available on the system and the native loader is selected a dialog box appears which shows how to install it 5 1 2 7 Neuralynx Support for Neuralynx files is available via a native loader By default all files in the same directory as the file loaded have the Neuralynx file naming conventions applied There is an option on the Neuralynx tab of the Tools Data Import Options dialog box that restricts Offline Sorter Software to loading only the selected file in which case there is only one channel available in Offline Sorter Software The Neuralynx loader supports all data types for spikes continuous data and events 5 1 2 8 NeuroExplorer Import for NeuroExplorer NEX files is available via a native loader and via the Neuroshare DLL which can be obtained from www NeuroExplorer com or www neuroshare org You can toggle between using the native loader or the Neuroshare loader using the NeuroExplorer tab of the Tools Data Import Options dialog box To use Neuroshare to load NeuroExplorer files you must download this DLL file and use the NeuroExplorer tab of the Tools Data Import Options dialog box to set the name and location of this DLL in the Neuroshare DLL Location section Only NeuroExplorer waveform variables are imported Each Unit gets its own channel in Offline Sorter Software For the native loader only NeuroExplorer Interval
91. the Units Automatically Recalculate Fit Tolerances after Each Change menu item or by clicking the Automatically Recalculate Fit Tolerances checkbox on the Templates tab in the Tools Options dialog box If the option to automatically recalculate the Fit Tolerances is not selected the initial Fit Tolerance given to a newly created unit can be specified via the Initial Template Fit Tolerance setting on the Templates tab of the View Options dialog box The Fit Tolerances for all existing units can be forcibly reset to a constant Fit Tolerance by clicking the Reset all units to have this Fit Tolerance checkbox and pressing OK Note that Offline Sorter Software can use the same Version 4 0 159 4 Spike Sorting Step by Step definition of Fit Tolerance that is used in the MAP system hardware by checking the Use MAP compatible Fit Tolerance checkbox see Section 6 5 2 Template Algorithm on page 300 When Offline Sorter Software calculates a Fit Tolerance for a unit it can use one of several different methods to do so By default the Fit Tolerance is calculated by finding the smallest Fit Tolerance value that would result in all waveforms currently assigned to the unit being sorted into that unit during a Template sort This corresponds to the Set to min max of waveforms radio button within the Fit Tolerance Calculation box in the Templates tab of the View Options dialog box Alternatively by clicking the Set to N times the s
92. the subsequent files are placed immediately in time after the end of the previous file The Create Time Segments for file extents checkbox will create Time Segments that correspond to the extents in time of the individual files loaded 228 Offline Sorter Offline Spike Sorting Software File Menu If desired the list of PL2 files along with the time gap specification and create Time Segments checkbox state can be saved into an OMF file for future use This OMF file can then be loaded directly during Step 1 above 3 Analyze and sort the data From this point Offline Sorter Software behaves exactly as if a single data file was loaded all functionality is available The following screenshot shows multiple files loaded into Offline Sorter Software with a gap of 5 seconds between files and each file demarcated with a Time Segment Se vom Out Zoomin lt m 576651 The Waveforms and Clusters views for example will show the data from all loaded files together Any operation performed in Offline Sorter Software for example sorting will be applied to the data from all files 4 Save the data After sorting the data can be Saved The Save filename dialog will prompt for a new OMF filename But the individual file names that are loaded into the multiple file session will be saved individually with filenames that are automatically generated by appending numbers to their original file names The saved OM
93. to be added or removed Waveforms can be un sorted or added to the current view from the Timeline view Right click on a waveform to select it and from the pop up menu select Unsort Waveform or Add Waveform to Selected Unit 4 6 3 3 Combining Units Sometimes what initially appeared to be multiple units is really a single unit The Units Combine Units menu selection enables several units to be selected and combine them into a single unit If multiple units are selected to combine in the Units view these units also appear as selected units in the Combine Units dialog Version 4 0 189 4 Spike Sorting Step by Step 4 6 3 4 Swapping Units For comparing units across files you can control which unit gets which unit label You can use the Units Swap Units menu selection to select two units to be swapped 4 6 3 5 Invalidating Units Occasionally an entire unit s worth of waveforms can be noise or other undesirable artifacts You can use the Units Invalidate All Waveforms in Selected Unit or Waveforms Invalidate All Waveforms in Selected Unit menu selection to invalidate all waveforms in the primary selected unit See Section 4 4 2 Invalidating Waveforms on page 129 4 6 4 Removing Spikes with Short Inter spike Intervals Most neurons have absolute refractory periods after generating an action potential Effective isolation of a single unit within a cluster should therefore have relatively few spikes with
94. to select cluster center 3 centers defined Naveform Detection Note The centroids cannot be moved or dragged To adjust the centroids positions remove all the centroids first and then select the new centroids Note When using the 3D Clusters view take care not to rotate the 3D viewpoint while selecting cluster centroids Any rotation or movement of the eyepoint performs a Clear All Centers operation so that the cluster centers must be re selected To remove the centroids select Clear Clusters from the right click menu 172 Offline Sorter Offline Spike Sorting Software Spike Sorting 4 5 8 2 Running the K Means Clustering Algorithm After the cluster centers have been specified start the algorithm To run the K Means clustering algorithm Click Find Units in the toolbar or select Find Units from the right click menu When the algorithm completes the points are sorted Version 4 0 173 4 Spike Sorting Step by Step As part of its operation the K Means algorithm removes the outliers from the clusters This is affected by the Outlier Thr parameter which can be adjusted by using the Control Grid To sort using the new Outlier Threshold rerun the K Means sorting algorithm 174 Offline Sorter Offline Spike Sorting Software Spike Sorting You can clean up the clustering results after the K Means sorting is complete by adding or removing waveforms from each unit e
95. view select Print to Windows Metafile 5 1 14 File Print All Sort Summaries The File Print All Sort Summaries command iterates through all channels and prints the Sort Summary for each channel to a single print job 5 1 15 File Send All Sort Summaries to PowerPoint The File Send All Sort Summaries to PowerPoint command iterates through all channels and exports the Sort Summary for each channel to a single PowerPoint file 5 1 16 File lt Recent Files gt The last 10 files that have been opened in Offline Sorter Software are available here for quick re selection 5 1 17 File Exit Exits Offline Sorter Software The user is prompted to save any unsaved work 236 Offline Sorter Offline Spike Sorting Software Edit Menu 5 2 Edit Menu The Edit menu contains operations such as undo redo reloading channels from disk and moving between recently viewed channels Edit View Select Waveforms Undo Add Unit Redo Add Unit Reload Channel from File cu Previous Channel in History CH Next Channel in History 5 2 1 Edit Undo Redo Offline Sorter Software maintains a standard undo redo stack for the current channel Most operations that affect the data can be undone and redone but some major operations cannot be undone The Undo Redo operations are also available via toolbar buttons and are mapped to the standard Ctrl Z and Ctrl Y hot keys 5 2 2 Edit Reload Channel from File
96. view displays the same time range as the time range that appears in the Timeline view and pressing the Zoom In or Zoom Out buttons or moving the time range scroll bar on the Rasters view is equivalent to doing the same in the Timeline view By default the Rasters view displays all channels that have spikes The number of channels displayed vertically can be changed by selecting Zoom In To Show Fewer Channels or Zoom Out To Show More Channels from the context menu or by pressing the I or o keys To scroll the display vertically move the mouse wheel or left click and drag the mouse in the label area on the left The display can be scrolled vertically by using the PAGE uP and Version 4 0 93 3 Views and Displays PAGE DOWN Or HOME and END keys You can also grab the dividing line between the labels and the tick marks and move it by left clicking and dragging the line The Rasters view reflects the Invalid w f setting in the Control Grid however it does not reflect the Waveforms view setting the Rasters view always shows the tick marks for all units and you can optionally show the unsorted waveforms by checking Show Spike Marks For Unsorted in the context menu By default each channel appears on a separate raster however if Show All Units for a Channel on One Raster is unchecked in the context menu a line for each sorted unit can be shown and possibly also the unsorted units which are denoted by a U postfix e g sig002U
97. views showing feature space can be used to select the template waveform To add a unit using the Template Sorting method The goal is to select a waveform to be used as a unit template 1 Press Add Unit button in the Clusters view 2 ove Wis Invalidate Wfs B Pos or hold down the ALT key and right click in the Clusters view and select the Add Unit menu command 2 Move the mouse pointer to the Clusters view eS The pointer changes to a drawing hand and the display is in a mode that selects the waveform closest to the pointer position Note For a discussion of waveform selection see Section 3 7 2 Clusters Views Common Functionality on page 54 The selected waveform appears in the Waveform display If Sel Wave is selected in the Waveform View entry in the Control Grid it only shows the selected waveform which can make it easier to select a template 3 When the desired waveform is selected shown with a box around it click the left mouse button A new unit is added and Template Sorting is carried out using all of the templates Version 4 0 157 4 Spike Sorting Step by Step To add a unit using the Template Sorting method using the Timeline View Alternately the bottom Timeline view can be used to select a template waveform 1 In the Timeline view right click and move the mouse to select the desired waveform Release the right mouse button Select Use Waveform As Templa
98. when the signal crosses the threshold in the negative direction find the first t such that signal t dt gt threshold and signal t lt threshold The time t then becomes the timestamp of the waveform The program stores the specified number of data points before t and the number of points after t in the extracted waveform that is used later for sorting See Section 4 2 6 Version 4 0 123 4 Spike Sorting Step by Step Specifying the Waveform Detection Options on page 120 to see how to set the waveform length and the number of prethreshold points e When the current waveform is extracted that is the specified number of data points after the threshold crossing is stored the program moves ahead in time by the amount specified by the Dead Time parameter see Section 4 2 6 Specifying the Waveform Detection Options on page 120 and then it starts looking for the next waveform This means that waveforms that cross the threshold less than the dead time after a detected spike are not detected To set the threshold use the threshold edit control in the Timeline view or drag the horizontal threshold line with the mouse Control Displays Center Selected Zoom 1 0 1 67 m Thr Cun 245 pV Detect Waveforms Options Detect Low cut Filter Butterworth 4 pole Z Cutoff Freq 250 Hz Apply Scroll Range 10 0000 0 0000 Zoom Out Zoomin lt Alternately dragging the vertical t
99. x ra Add Unit Find Units Delete Unit Add Wfs Remove Wfs Invalidate Wfs Adding unit Draw a line around a cluster Whether or not this text is displayed is controlled by the Show Hints and Show Statistics menu commands in the right click menu If the Show Ellipses or Show Ellipsoids on the 3D Clusters view menu command is selected ellipses ellipsoids for each unit are shown on the display The ellipses are calculated by finding the standard deviations along the principal axes The ellipse or ellipsoid is scaled by the number of standard deviations specified by the Outlier Threshold in the Control Grid 3 7 2 7 Colors To set the colors assigned to each unit in the Clusters views and Units view select Tools Options menu command and select the Colors tab 3 7 2 8 Point Size To set the point size in the Clusters views select the Display tab under the Tools Options dialog box 3 7 2 9 Automatic Cluster Redraw To update the Clusters view immediately when the Slice 1 Slice 2 Slice 3 or Slice 4 values change select the Redraw Cluster Display Immediately when Slice Position Changes in the Display tab under the Tools Options dialog box Version 4 0 59 3 Views and Displays 3 7 2 10 Zooming and Panning Both of the Clusters views support similar mechanisms to zoom and pan in the other display Hold down the right mouse button and move the mouse vertically to zoom the display in and out To pan the d
100. 0 24975 250 pv SPK 61 62 63 64 l 04315 250 pV WB 1 2 3 4 i l O 0 pv WB 5 6 7 8 i A WwB 910 1112 2 0 0 pv WB13 1415 16 7i 00 pv WB 17 18 19 20 i i O 0 pv WB 21 22 23 24 i a 0 0 pv WB 25 26 27 28 2 00 pv WB 29 30 31 32 a 00 pv WB 33 34 35 36 i O 0 pv WB 37 38 39 40 A WB 41 42 43 44 i 2 00 pv WIB AB AR A AD NIN wh pa ee ee ee ee E E E e The Channel Parameters view can contain different columns depending on the file type currently loaded Name displays the channel name Channels can be renamed by clicking in this column and typing a new name Sig ch for PLX files only shows the channel number on the SIG board this is associated with this channel Raw Srec Ch shows the Source names and within Source raw channel numbers associated with the Offline Sorter channel Note that for stereotrode tetrode data there may be more than one raw channel associated with a single Offline Sorter channel as shown in the screenshot If there is an associated Source involved both Sources are displayed Freq shows the sampling frequency for this channel If continuous data was upsampled both the original and the upsampled frequency is shown Spk Gain indicates the overall gain setting for the spike signal path taking into account all preamp and final amplification gains Cont Gain indicates the overall gain setting for the continuous data path taking into account all preamp and final a
101. 00 ISIHistMax ms 100 Use Range E Range from s 0 000 Range to s 550 541 Invalid wif Show When the threshold marker line appears you can click and drag the line up or down to establish a new threshold As soon as the drag is completed the waveforms are re thresholded You can undo the effects of re thresholding the data either with the Undo command or with the Waveforms Mark All Waveforms Valid menu selection 5 8 6 Tools Interval Selection A valid reason to mark waveforms invalid is that they are simply not of interest For example if the experimental animal was not exhibiting interesting behavior during certain time intervals you can invalidate the waveforms collected from that interval simply to get them out of the way Offline Sorter Software contains a tool that enables the selection of waveforms within some interval in time around an event or between events in the data file Or more accurately it enables one to invalidate those waveforms that do not meet the 264 Offline Sorter Offline Spike Sorting Software Tools Menu selection criteria those that are not in the interval Select Tools Interval Selection from the main menu to display this window F Interval Based Waveform Invalidation This tool will allow you to keep or invalidate waveforms based on where they occur relative to Events in the file or based on their times You must specify the waveforms that you wish to kee
102. 00 0000 65 128 Y 16 2500 00 129 192 Y 2500 0000 Version 4 0 279 6 Reference Topics After loading this data file the Channels view shows a total of 256 4x64 channels A portion of the Channels view with Offline Sorter Channel 129 selected is shown in this screenshot S Bae Sage want 0 459500 Each Source corresponds to a contiguous range of Offline Sorter Channels these channel ranges are shown for each Source in the Chs column in the Sources view Each Source also represents a range of raw channel numbers in the data file this range is shown in the Raw Chs Column This range of raw channel numbers in general does not match the range of Offline Sorter Channels for a Source However the Source s corresponding to an Offline Sorter Channel will always have matching trodality single electrode stereotrode tetrode Offline Sorter Channels will always corresponding single electrode stereotrode tetrode Sources 280 Offline Sorter Offline Spike Sorting Software Details of Channels and Sources As mentioned above the Channel Parameters view will show which Source and raw channel number are associated with each Offline Sorter Channel ae ed ee Ed Preamp Ga In this example Offline Sorter Channel 129 corresponds to raw channel number 65 in the SPKC Source A single Offline Sorter Channel may be associated with both a spike and a continuous Source P
103. 202 Offline Sorter Offline Spike Sorting Software Examining the Sorting 4 7 2 2 Statistics Feedback While Sorting On the 2D and 3D Clusters views there are mechanisms that can be used to get immediate feedback on the sorting quality while performing sorting It is sometimes useful to know immediately if an action e g removing outliers from a cluster helped or hurt the sorting quality If the action hurt the sort quality it can immediately be undone by using the Edit Undo menu selection There are two similar ways to display the sort quality statistics on the Clusters views The descriptions follow You can display the absolute values of all Sorting Quality Statistics which are detailed on page 306 in a white bar along the bottom of the view by selecting Show Statistics from the right click menu The abbreviations appear in this line are e p the p value from the multivariate ANOVA e J3 the J3 statistic e PsF the Pseudo F statistic e DB the Davies Bouldin statistic e Dn the Dunn stastistic e LRatio the worst L Ratio statistic for any unit e IsoDist the worst Isolation Distance statistics for any unit There are several available Sorting Quality Statistics that can be chosen for display at the top of the view in a green or red bar by selecting Show Cluster Quality from the right click menu You can choose the Sorting Quality Statistics to display in the top bar from the Sorting Quality Statistic to use on the
104. 3 2D Pseudo F 2D Davies Bouldin 2D Dunn 2D p 3D F 3D Remove All What File to Store the Exported Data in File E PlexonData Plexon pls testwaveforms sorted txt Which Channels Current Channel only C All Channels into one file Chosen Number of Units Number of Waveforms Browse Append if existing Version 4 0 209 4 Spike Sorting Step by Step To perform an export 1 Select the Format for the exported information either text file MATLAB file if MATLAB is available on the computer or Microsoft Excel if available on the computer For text export choose a delimiter to use between values Delimiter choices include comma comma and space space or semicolon For Excel and text export check Export a Row of Column Headers to get a header line of text that labels each of the columns This makes reading the exported file easier Select Which Channels to export If a current channel is selected in Offline Sorter Software when the File Export Per Channel Data menu selection was selected the default is the Current Channel only You can also export all channels to a single file Select What to Export for Each Channel This enables selection what items appear in each column for each exported row of data The Available list box on the left shows all the items that can be exported and the Chosen list box on the right shows those that have been selected
105. 3D cluster space The Null hypothesis is All unit clusters actually have the same underlying distribution in 2D or 3D space and have the same mean i e do not represent distinct spike waveforms The p value is The probability of rejecting the null hypothesis that all clusters are actually the same if it were actually true Conclusion The smaller the p value the more confident you can be in rejecting the null hypothesis and concluding that the clusters are in fact distinct and represent different units 6 6 2 J3 J3 is a non parametric measure of the quality of sorting i e no normality assumptions are involved J3 J2 J1 where J1 XXE f u i m u is a measure of the average distance in feature space between points in a cluster f from their center m E x represents the Euclidean distance squared x x The summations are over units u and over feature vectors points in each unit i J2 XN u E m u m is a measure of the average distance between unit clusters The summation is over units u N u is the number of points in unit u E x represents the Euclidean distance squared m u is the cluster center for unit u and m is the grand center of all points in all units So J3 takes on a maximum value for compact well separated clusters References Wheeler Bruce C Automatic Discrimination of Single Units in Methods for Neural Ensemble Recordings ed by Nicolelis M CRC Press Boca Raton 1999 6 6 3 Pseud
106. 80641 8046 25 Davies Bouldin 0 24361 0 35362 Dunn 1 85999 1 01824 LRatio 13 9334 14 225 Iso Dist 17 5808 19 0243 Co Per Unit Sort Quality Iso Dist 3D 74 010461 i 62 696920 25 815006 119 024344 48 702321 L Ratio 2D 0 114751 0 409427 13 933393 2 799314 lt 11 097177 Iso Dist 2D 168 313518 59 556868 23 489032 17 580806 34 813274 L Ratio 3D 0 254181 0 426818 14 224962 4 161291 11 676796 Per Unit Statistics Counts lt ISI lt 100 Center 2D Sigmas 2D Center 3D Sigmas 3D 2156 0 0000 12138 4108 2316 2038 9675 1475 6 12138 4108 23163 0403 3 3184 9327 1775 6566 1388 74391 0 0000 1888 8704 2611 2351 4002 1415 2 1888 8704 26119 5217 11 2860 8655 2208 1715 1403 285 10 0000 1930 0041 7591 1849 1777 1133 4 1930 0041 7591 9530 170 1976 2594 1792 1244 1133 334 10 2994 7311 5544 4240 2 2049 9533 1697 3 7311 5544 4240 2443 15 7 2819 1779 2036 6154 1524 284 0 0000 25997 3163 2709 2411 8216 1539 5 25997 3163 27093 9377 61 4842 8716 2007 5608 1487 Normality Statistics Counts Skew 2D Kurtosis 2D Skew 3D Kurtosis 3D Unit a Unit d All Valid WFs Unsorted WFs Unit b Unit c Unit e i 7748 2924 2156 1391 1285 284 334 0 2613 1 1740 lt 0 1828 0 6601 0 0525 0 0371 0 0697 0 1217 lt 0 0730 0 0546 0 2308 0 0663 20 0523 0 1115 0 2324 1 1305
107. F file name will then contain these modified output file names in its list of PL2 file Thus loading the output OMF file will load all the newly Saved individual PL2 files together into a multiple file session This behavior reinforces the illusion that the OMF file contains the data saving a new OMF file allows loading that OMF in the future and seeing the results of all work performed on the data Note As of Offline Sorter v4 0 the equivalent of the Export to New PL2 functionality is not available for OMF files that is for multiple file sessions This means that for example if waveform extraction from continuous data is performed during a multiple file session the results cannot be saved into new PL2 files The results can however be saved into a single plx file that contains the data from all files 5 1 4 File Close This command closes any open file and puts Offline Sorter Software into the same state that it is in when it is first launched It is generally not necessary to select this menu item simply opening another data file will close the previously open file 5 1 5 File Save If Offline Sorter Software is used to resort spikes from a file with previously sorted waveform spike data e g a Plexon PLX or PL2 data file or Datawave the resorted waveforms and timestamps can be saved into another file of the same format All records in the original file are preserved in the output file except that Versi
108. File Export to New PL2 menu option possibly with a numeric suffix Note that this option is only available if the file being processed is a PL2 file e ExportToNex or SaveToNex saves the timestamps etc to a NEX file possibly with a numeric suffix added on to the original filename e SaveTpl saves a sorting template which is specified by the Set TplFileName command e SortUsingTpl applies a sorting template which is specified by the Set TplFileName command e InvalidateArtifacts applies cross channel artifact removal the current file using parameters specified by the Set ArtifactXXX commands e InvalidateArtifactsAfter applies cross channel artifact removal to the current file after the ForEachChannel commands are processed See the Notes section below ExportWaveformInfo writes out selectable information about each waveform in all channels to text files a MATLAB file or an Excel spreadsheet The information to be written and other controlling parameters must be specified in the File Export Per Waveform Data dialog e ExportUnitInfo writes out selectable information about each unit in all channels to either text files a MATLAB file or an Excel spreadsheet The information to be written and other controlling parameters must be specified in the File Export Per Unit Data dialog e ExportChannellnfo writes out selectable information about each channel to either a text file a MATLAB file or an Ex
109. J Neurosci Meth 12 95 111 1984 Wheeler B C Automatic Discrimination of Single Units Methods for Neural Ensemble Recordings ed by M Nicolelis CRC Press Boca Raton 61 77 1999 Version 4 0 13 2 System Overview Wheeler B C and Heetderks W J A comparison of techniques for classification of multiple neural signals IEEE Trans Biomed Eng 29 752 759 1982 2 4 Offline Sorter Outputs Offline Sorter Software provides a variety of ways to view and save the results of spike sorting The Offline Sorter File menu includes the following actions 2 4 1 Printing Results The 2D and 3D Clusters views can be printed as well as the Waveform Display and the various grid based views Section 3 14 Sorting Summary View on page 84 provides a convenient summary of the sorted units on a channel and can be printed or exported to a Microsoft PowerPoint or an Adobe PDF file For more information see Section 5 1 13 File Print Print Preview Print Setup on page 236 and Section 5 1 14 File Print All Sort Summaries on page 236 2 4 2 Saving Results For PLX PL2 Datawave and Neuralynx data files Offline Sorter Software can save the unit assignments for each spike back into an output file of the same type Offline Sorter Software preserves all records from the original file in the output file except that it modifies the unit assignments for spike records to reflect the sorting that has be
110. Last Used Settings F For View wf Options Editor to use for Batch Command Files notepad exe m File Save Behavior Preserve Original prompt for new filename defaults to filename On X Default Directory for Output Files Same as Input File Always use C PlexonData The options that pertain to similar features or functions are grouped together into tabs The individual options are described in the sections describing the feature or function that each option pertains to these descriptions will not be repeated in this section Generally performing a text search within this user guide for the option label on the dialog will find the description of that option 270 Offline Sorter Offline Spike Sorting Software Tools Menu 5 8 15 Tools Data Import Options The options that control the behavior of the loader for the different data file formats are grouped together as tabs in the Tools Data Import Options dialog box You can open the dialog box from the menu or click or and then on the General tab click Data Import Options The Data Import Options dialog box opens E Data Import Options Multi Channel Systems Nein Nene tamar BC Beams Plexon Alpha Omega CED Spike 2 Cyberkinetics Ignore Spike WaveformFreg from PLX File Header Invalidate Incomplete Stereotrode and Tetrode Waveforms V Compensate for gain differences within Stereotrode
111. NOVA and the other statistics calculations The next lines contain the ANOVA F and p values as explained in Section 6 6 Sorting Quality Statistics on page 306 The next section contains a table with a row per unit plus entries for all waveforms and for unsorted waveforms The columns are e Counts The total number of waveforms in the All Valid WFs row the number of unsorted unassigned waveforms in the Unsorted WFs row and the number of waveforms in each unit e RMSE the summed root mean squared for all waveforms in the category This is a sample by sample summation of the deviation from the average waveform summed across all waveforms This sum is then divided by the total number of ticks then the square root is taken RMSE 4 ZZ v w t ave t Nw Np where the sums run over N waveforms w and N ticks t in each waveform v w t is the waveform amplitude at tick t for waveform w avg t is the average amplitude of all waveforms at tick t Thus this number is smaller for compact clusters The Plexon MAP system calculates a similar summed squared difference for the real time online Template Sorting unit assignment algorithm ISI Percentage lists the percentage of waveforms that occur within the Refractory ISI period as set by from the Control Grid of each other Large percentages here for a unit can indicate that errors have been made in the sorting of that unit The next section to the right displ
112. None in the illustration that follows 2 Select the channel from the drop list orin the Channels view of the Info Grid Views double click anywhere on the row of the desired channel SPKO1 0 591 Lem Name Samples Waveforms Unsorted Unita Unitb Unte eT 23 145172 51 f 152 144 149 148 138 0 0 0 0 e MUM 0 0 n The main views in Offline Sorter Software then show the channel s data 3 To move quickly to the next or previous channel in the list of channels click Select Next Channel or Select Previous Channel The following toolbar buttons can also be used ee 126 Offline Sorter Offline Spike Sorting Software Preparation For Sorting 4 Offline Sorter Software maintains a history of previously opened channels To revisit a previously opened channel click Edit Previous Channel in History or Edit Next Channel in History The following toolbar buttons can also be used Ex th 4 4 Preparation For Sorting 4 4 1 Data Inspection After the segmented waveforms spikes are acquired they are displayed in the Waveforms view and the Clusters views Offline Sorter Software allows the user to quickly get an overview of the data 4 4 1 1 Waveforms View The Waveforms view shows the superimposed waveforms aligned at the threshold crossing times TIP Controlling Which Waveforms Are Displayed The scroll bar at the bottom of the Timeline View use th
113. OTIOINS sorosiane annen aarne aE 237 Version 4 0 217 5 Menu Options see PIE PMS EA AN A AE EA E A AT A A 238 E E E T A E E E A A E A 240 5 4 1 Select Next Channel Previous Channel ccccccccccesssceeeeeeenteeeesescneeeeseeeaees 240 5 4 2 Select Next Unit Previous Unii osaisniissssnsrnsannainnnna 240 5 4 3 Select Next Waveform Previous Waveform sscccceessseeeeeesessaeeeessenaaeeeeeseaes 240 5 4 4 Select Next Segment Previous Segment sssesseesseeeseesesssenssrrrssrnsssrnssnnent 241 5 4 5 Select Feature Space DOWNS wisccicisccicsecdcicanmedcbenaduenadvanenedciesesanicdiorameeaiiads 241 ee WV el OMS WIG RIO 445 scxncv ensue ieee ieee areas NA ana 241 5 5 1 Waveforms View Only Currently Selected Unit s ccccceeeseeeeeeeeeeeneeeeees 242 5 5 2 Waveforms View Only Unsorted Waveforms cccccseeecereeeeeeeseeeeeeeeaeeeenes 243 5 5 3 Waveforms View Only Selected Time Segment ccsccceceecseeeeeeesenneeeeeseeaes 243 5 5 4 Waveforms Create Time Segments sorsien aiani 243 5 5 5 Waveforms Use Only Currently Visible Waveforms c cccccecsseeeeeesstteeeeeeeaes 245 5 5 6 Waveforms Use Only Unsorted WavaforMS sisirin 246 5 5 7 Waveiorms invalidate WaVGIONMNS sns 246 5 5 8 Waveforms Invalidate All Except Selected Waveforms cccsscceeseesstteeeeees 246 5 5 9 Waveforms Mark All Waveforms AS Valid 0 csccceeeeesecceeeeeesee
114. Offline Sorter Software can read the TPL file and use the information to resort the current file For more information see Section 4 8 4 2 Sort Using Saved Templates on page 214 2 4 3 3 Sort Using NEX file Offline Sorter Software can read a previously saved NEX file and apply the sorting embodied in the NEX file to the currently loaded file This only makes sense if the NEX file was saved from the same file as is currently loaded For more information see Section 5 6 9 Sort Sort Using NEX File on page 255 Version 4 0 15 2 System Overview 2 4 3 4 Execute Batch Command File Offline Sorter Software has a batch processing facility that can perform sorting actions based on simple commands read from a text file This facility is useful for automatically sorting large files which can be time consuming For more information see Section 6 9 Batch File Processing on page 313 2 5 Terminology In the discussion below continuous and digital refer to types of data 2 5 1 Spike Waveform Segments The term spike waveform segments describes the waveform segments around threshold crossings These are more commonly called simply spikes or waveforms 2 5 2 Continuous In general continuous is used throughout this manual to refer to any signal that was recorded continuously for as long as the data acquisition switch is turned on in Sort Client or the OmniPlex D System Continuous excludes spike waveform
115. Original Normalized Comment J3 J3 Units dividing by the number of units PsuedoF log PseudoF taking the logarithm Davies Bouldin Davies Bouldin flipping the sign Dunn Dunn no change p Value log p Value taking the negative logarithm LRatio LRatio flipping the sign IsoDist IsoDist no change For the normalized sort quality metrics larger numbers are always better and the range of values is generally within an order of magnitude or so of unity To enable the calculation and use of the Normalized version of the sort quality metrics check the Normalize checkbox Note Other settings in Offline Sorter Software can also affect the results of the scan For example the Include unsorted waveforms as a unit in the statistics calculations in the Tools Options Stats tab affects the Sorting Quality Statistic values The available scan types are Note Scan Parzen Radius Multiplier for Valley Seeking This scan type steps the Parzen Mult parameter as can be set via the Control Grid for the Automatic Clustering using Valley Seeking Method page 176 sorting algorithm over the range specified by the Start End and Step By values re running the sorting each time The Limit Number of Waveforms used in Valley Seeking Sorting setting in the Tools Options Sort tab can affect the clustering Scan D O F Multiplier for T Dist E M This scan type runs the Automatic Clustering using T Distribution E M Method page 178 sorti
116. Overlap Templates just as if they were normal spike waveforms and plot the points in feature space on the Clusters Views This then indicates where waveforms that match each Overlap Template would appear in the Clusters views When activated the Overlap Templates are plotted as large multicolored blocks connected by multicolored lines Clusters 102 Offline Sorter Offline Spike Sorting Software Overlaps Analysis View 3D Clusters The Overlap Templates generated by incrementally offsetting different unit templates generally form interesting patterns in feature space as the method of generating the Overlap Templates by marching through the relative offsets has the effect of pushing waveforms along trajectories in feature space This can give insight into whether unsorted and or noise waveforms are actually due to overlaps When an unsorted waveform matches to within the Tolerance setting one of the Overlap Templates it is rendered in yellow in the grid cell corresponding to the Overlap Template that it matched The yellow text in the lower left corner of the grid cell shows the number of unsorted waveforms that matched that Overlap Template The total number of unsorted waveforms that matched any Overlap Template in any permutation of units are shown on the left under Matched Overlaps These are the overlapping waveforms that will be rescued when the Rescue Overlaps button is pressed Version 4 0 103 3 Views
117. Rng Offline Sorter Software can be configured to analyze and sort waveforms using only a subset of the available samples within spike waveform segments The indexes of the samples being used the sort range is shown in this column for spike Sources only If Offline Sorter Software is using the full spike waveform segments then the sort range will be 1 NPW Note The currently selected spike Source is designated with an S gt in the first column and the currently selected continuous Source is designed with a C gt See Section 6 1 Details of Channels and Sources on page 6 277 28 Offline Sorter Offline Spike Sorting Software Info Grid Views 3 2 2 Channels View E gt gt 1 Ex ES Ea Ea Es Eas Bs EA Era EEA 14 ES 20 21 Ea _ 23 pte Sn wolfe re Uae owe one Jooo COCO OOO OOOO OOOO oOo SD The Channels view offers a quick overview of the contents and the sorting status of a file The Channels view contains a row for each channel in the file The columns that display in the Channels view depend upon the file type that is open For data files with timestamped spikes e g PLX or PL2 files as pictured earlier the Channels view contains the following columns Name displays the name of the channel Samples indicates the number of data points for continuous channels if the channel has only spike waveforms the number of data points can be 0 Waveforms
118. Section 3 13 Feature vs Feature View on page 80 Sorting Summary View The Sorting Summary view displays a concise printable summary of the sorting on a channel The Sorting Summary view can also be exported to PowerPoint For more information see Section 3 14 Sorting Summary View on page 84 PCA Results View The PCA Results view graphically displays the information that can be found on the PCA view of the Info Grid Views Each of the PCA eigenvectors can be plotted along with a bar graph of the eigenvalue magnitude For more information see Section 3 15 PCA Results View on page 88 ISI Histograms View The ISI Histograms view shows the same ISI Histograms that are plotted in the Unit Display see below but the view can be printed and it offers several other display options such as log scaling of the axes For more information see Section 3 16 ISI Histograms View on page 90 Cross Correlograms View The Cross Correlograms view shows a matrix of cross correlation plots between all of the units in the current channel For more information see Section 3 17 Cross Correlograms View on page 91 24 Offline Sorter Offline Spike Sorting Software Info Grid Views Rasters View The Rasters view shows Raster Plots of the spike and event times for all channels For more information see Section 3 18 Rasters View on page 92 Scan Graph View The Scan Graph view displays a graphical view of
119. Software Overview 3 1 Overview All available views within Offline Sorter Software have corresponding menu items under the View main menu entry and many of the common views also have corresponding toolbar buttons Selecting a menu item or pressing the corresponding toolbar button will create and show the View if it is not already showing Control Grid Units Waveforms Timeline 2D Clusters 3D Clusters Surface Clusters vs Time Templates vs Time Waveform Inspection Waveform Detection Continuous Frequency Spectrum Feature vs Feature Sorting Summary PCA Results ISI Histograms Cross Correlograms Rasters Scan Graph Sort Quality vs Time Segment Graph Threshold Scan Graph Overlaps Analysis Waveforms Legacy Animate Note The Animate menu items the last option in the menu list above are not actually views Instead they allow you to play back the data in a file in time order See Section 5 3 2 View Animate on page 5 238 Following is a brief description of each of the available views and a link to more detailed information about each view Version 4 0 21 3 Views and Displays Info Grid Views The Info Grid Views are a collection of spreadsheet like informational views that are available under the View Info Grid Views sub menu The available Info Grid Views are listed below See Section 3 2 Info Grid Views on page 25 Control Grid Units Waveforms Timeline 2D Cl
120. Spike Sorting Software Offline Sorter Screen Layout 2 6 Offline Sorter Screen Layout Offline Sorter Software has a highly customizable screen layout By default it contains the following screen elements Per View Toolbars Waveforms View 2D Clusters View Tabb a a ed Views ae Tee me Lx PB a p Toolbars _ ERN S ELEAN Control z Grid 7 gt t EER CEH CH3 Units a View Ptn Chaters M030 hetes 20a dace Pesk integr a CGT RT V San OE TTT Timeline eee UAE PA ote oF THIET E T Status Bar 2 6 1 Toolbars Frequently used menu items have equivalent buttons on the toolbars Move the mouse pointer over a toolbar button to open a tooltip window that describes the function of that button The toolbars can be moved and docked to the top bottom or sides of the screen The contents of the toolbars can be rearranged and customized by using the Toolbars tab under Window Customize menu item TIP Keyboard Bindings Common operations may be conveniently and quickly performed by binding a keyboard shortcut to any function available from the menus or toolbars Use Keyboard tab under Window Customize menu item 2 6 2 Status Bar At the bottom of the screen is a status bar Tool tips for the currently selected toolbar or menu item appear at the left edge of the status bar When Offline Sorter Software is performing a time consuming operation a p
121. Templates button in Templates tab of the Info Grid Views Be aware that disabling automatic recalculation of unit templates can lead to confusing displays because sometimes unit templates may not exist until they are explicitly calculated and will show up as flat lines for example after performing a Valley Seeking sort Note The unsorted unit is not affected by the automatic recalculation setting The unit template for the unsorted pseudo unit is always recalculated after any sorting change 158 Offline Sorter Offline Spike Sorting Software Spike Sorting 7 Options Colors Display Printing Opti Active Features Cache General Feature Calc Sort Templates Stats Refractory Period Unit Templates and Fit Tolerances V Automatically Recalculate Unit Templates Z Automatically Recalculate Fit Tolerances Fit Tolerance Calculation 5 Setto 1 times the standard deviation of wavefoms F Use MAD Set to min max of waveforms Initial Template Fit Tolerance 70 E Reset all units to have this Fit Tolerance V Use MAP compatible Fit Tolerance Adaptive Templates Weight 0 01 Threshold for Template Update 20 By default recalculating the Fit Tolerances is only done when the user explicitly requests it by selecting Units Calculate Fit Tolerances from Existing Units but it can also be done automatically after any change to the sorting is made by clicking
122. The Sort Start and Sort End times are represented by vertical lines in the Waveforms view see the arrows in the preceding illustration Small arrows on the top and bottom of the lines indicate whether it s the start or end In the previous example the total waveform length is 1000 msec the Sort Start time is 150 msec and the Sort End time is 650 msec 142 Offline Sorter Offline Spike Sorting Software Spike Sorting Note For stereotrode and tetrode data the sort range is specified per electrode not for the concatenated waveform 4 4 6 Selecting Features to Use For Sorting For those sorting methods that use feature space i e all methods except Waveform Crossing and Templates one must first decide which feature space to work in One does this by selecting particular features for each of the 2 for the 2D Clusters view or 3 for the 3D Clusters view axes in feature space See Section 6 4 Features Available for Sorting on page 294 Offline Sorter Software does all spike sorting and calculates all statistics in the feature space selected so the objective is to find a combination of features that gives maximal cluster separation The description in Section 3 13 Feature vs Feature View on page 80 can be useful in selecting which features to use First select either the 2D Clusters view or the 3D Clusters view using the View menu command or the toolbar buttons a 30 Then select features for the X Y and
123. This command will reload the currently loaded channel from that last saved to the file This is useful to provide a fresh start for a channel where something has gone wrong with the sorting or processing 5 2 3 Edit Previous Channel in History Next Channel in History Offline Sorter Software maintains a stack of the channels previously opened These operations can navigate forwards and backwards through this list of channels 5 3 View Menu 5 3 1 List of View Menu Options The majority of the View menu contains entries to open and display each of the major views in Offline Sorter Software Selecting menu items in the View menu causes the view to be opened on the screen if it is not already open See Chapter 3 Views and Displays for a full list and descriptions of these views The descriptions of the individual views will not be repeated here The View Animate function is described below Version 4 0 237 5 Menu Options Info Grid Views gt Control Grid Units Waveforms Timeline 2D Clusters 3D Clusters Surface Clusters vs Time Templates vs Time Waveform Inspection Waveform Detection Continuous Frequency Spectrum Feature vs Feature Sorting Summary PCA Results ISI Histograms Cross Correlograms Rasters Scan Graph Sort Quality vs Time Segment Graph Threshold Scan Graph Overlaps Analysis Waveforms Legacy Animate 5 3 2 View Animate A useful technique with Offline Sor
124. Unsorted Waveforms tool to assign the waveforms to one of the clusters the Valley Seeking algorithm creates The outlier threshold specified in the Control Grid also affects how these skipped points are assigned to units Finally you can use the Tools Remove Outliers 176 Offline Sorter Offline Spike Sorting Software Spike Sorting menu selection to optionally run following the sort in order to remove outliers using the outlier threshold value For more information see Section 4 6 3 1 Automatic Outlier Removal Tool on page 187 To run the Valley Seeking clustering program Click Find Units in the toolbar or select Find Units from the right click menu or select Sort Sort Current Channel Run Valley Seeking Sorting The algorithm attempts to determine the correct number of clusters and assign the waveforms to those clusters Note Select Sort Stop Sorting to stop the algorithm after the next iteration completes The algorithm runs until convergence unless it is cancelled or until a maximum number of iterations completes There is a parameter in the Control Grid labelled Parzen Mult that appears when selecting the Valley Seeking sorting method This parameter controls the behavior of the algorithm see Section 6 5 7 Valley Seeking Algorithm on page 304 for more details You can clean up the automatic clustering results after sorting completes by adding or removing waveforms from each un
125. You can invoke this tool automatically following a Valley Seeking automatic sort This is useful if the Limit Number of Waveforms used in Valley Seeking Algorithm option is used as this can leave points unassigned that clearly belong to units You can set these options in the Sort tab of the Tools Options dialog Note Temporarily setting Outlier Thr to a large number in the Control Grid before running this tool can force the algorithm to assign all unsorted points to a cluster regardless of the distance of the point from the nearest cluster center 5 8 10 Tools Invalidate Cross Channel Artifacts Invokes the outlier removal algorithm described in Section 4 4 2 4 Automatically Invalidating Artifacts on page 135 5 8 11 Tools Invalidate High Amplitude Artifacts Shows the following dialog Version 4 0 267 5 Menu Options 7 Invalidate High Amplitude Artifacts This tool will invalidate artifacts that exceed a certain fixed amplitude with the option to invalidate subsequent spikes that occur within a time period Which Channels Current Channel only All Channels in Spike Source SPK Chs 1 64 Amplitude threshold for artifacts of ADC Range microVolts ADC Counts 90 00 17999 88 29491 After an artifact is detected invalidate all spikes for 0 milliseconds afterwards Ca cone This tool allows the user to set an amplitude threshold for artifacts and invalidate any waveform that cross
126. ain 1000 Continuous Full Scale Voltage at Probe mV 2 500 lt lt lt Peak Voltage at ADC Input mv 2500 000 A D Resolution bits 16 Voltage Resolution at Probe mV bit 0 000076 Preamp Gain 1 TotalGain 1000 Set Gains on a Range of Channels LC x _ 4 P iil all gt See descriptions of the gain related items under Section 3 2 3 Channel Parameters View on page 30 and Section 3 2 4 File Summary View on page 32 One situation in which having misrepresented gains can be problematic is for waveform extraction It is recommended that before waveform extraction the Adjust Gains dialog should be inspected for one or more channels If the gain settings for the spike and continuous data are such that the Full Scale Voltage at Probe value for spikes is significantly different that the value for continuous data the result of the extraction could be sub optimal in one of two ways e Ifthe Full Scale Voltage at Probe value is much greater for spikes than it is for continuous data then the extracted spikes will be too small That is the largest extracted spike will use only a small portion of the dynamic range available for spike waveforms This means the resolution and detail is lost e If the Full Scale Voltage at Probe is much less for spikes than it is for continuous data then the extracted spikes will be too large That is the extracted spikes are likely to exceed the maximum r
127. ality cceeee 54 Version 4 0 19 3 Views and Displays Be SNIE VEN aana REA 69 20 MISS vs Timne View ssoi nioena siara 71 3 10 Waveform Inspection WOW ssccccssieiceiersiicccereieioieecesuinduedersyiunes 73 3 11 Waveform Detection ViIGW ccsccviesiicneiccnsoneirensesniivenaionnevedernine 74 SIA Threshold CU CUIPATIGD neria 75 3 11 2 Adjusting the Threshold for Waveform Detection 76 211 3 Changing mhe DSTO uisi 76 3 11 4 Stereotrode and Tetrode Waveform Detection 78 3 12 Continuous Frequency Spectrum View sses 80 3 19 Feature ys Feature VIOW accirascuiccsciccyisdedieceialeecsesuincmbersvicons 80 3 14 Soring Summary ViN sosia atimerneaes 84 3 14 1 Customizing the Sorting Summary ssssssseseseeeeseeeee 87 3 14 2 O tput to FowerP ont seriinin 88 3 14 3 Outoutting for All Channels siccccccsscnicccsseetontanieneseconmce 88 51S POA Results VEW orsin rana 88 So CU USI VEW sanirane aana 90 3 17 Cross Correlograms View occccinceriiccteasesccsctecaiaiconesacevnastcameracse 91 2 19 Raster YOW annenin a 92 SIS ocan GEADA VIEW oanwiisd 94 3 20 Sort Quality vs Time Segment Graph VIEW sssssssseeeeseeeeeeen 97 3AT Threshold Sean Graph cide scares ceca nanne 97 B22 Overlaps Analysis VIEW eusiininniamnnnnaii 99 3 22 1 Overlaps Analysis Operation cccceeeseeeseeeeeenees 101 3 22 2 Rescuing VON AIG osc cipuiaaccdraiitiedintaaticndesvlonebeeias 104 20 Offline Sorter Offline Spike Sorting
128. and Displays Tolerance 70 Template Sort Units J Minimum Overlap Run Overlaps Analysis Unit Permutations 6 Overlap Templates 45 Matched Overlaps 89 Showing Units a c lt Prev Next gt V Show on Clusters Views v Show Unit Shift Text Rescue Overlaps Another feature of the display is a red unit designation that can appear for some Overlap Templates on the bottom e g the units a and b that are displayed in the Overlap Templates labeled 0 22 and 0 7 in the above screenshot This display is actually a warning of a potential problem It is displayed whenever a unit template matches to within the Tolerance any Overlap Template That is the Overlap Template that was constructed from superimposing shifted versions of the unit templates ended up matching one of the unit templates perhaps one of the unit templates that was used in the construction of the Overlap Template or perhaps a different unit entirely This might indicate a mistake in the original sorting of the units used to create the Overlap Templates perhaps the units shown in red was actually composed of overlapping waveforms from other units More often it is an indication that Tolerance value is set too high 3 22 2 Rescuing Overlaps When the Rescue Overlaps button is pressed Offline Sorter Software will effectively split each overlapping waveform as determined above the waveforms rendered in yellow into two
129. and Tetrode channels Maximum size in words of continuous data 120 blocks forthe Export As New PLX operation PLX Slow Channel Preamp Gain 500 f not specified in file Strobed Events E Create Separate Events for All Strobed Codes Name Generation Code Representation Prefx SE Code Format decimal Code Length characters 5 Sort Range Use Sort Start and End imported from the file Always reset Sort Start and End to the entire waveform Always set Sort Start and End to in microseconds enter 0 Start 0 End 0 for end of waveform oe Cea The options that pertain to each different file format are grouped together into tabs and some options that affect multiple file formats are placed in a General tab The individual options are described in the sections describing the import of data from each file format e g see Section 5 1 File Menu on page 220 These descriptions will not be repeated in this section Generally performing a text Version 4 0 271 5 Menu Options search within this manual for the option label on the dialog will find the description of that option 5 9 Window Menu The Window menu contains entries for controlling the appearance and layout of the windows within Offline Sorter Software Window Run Help Theme Layout Customize See Appendix A 3 1 Window Menu on page A 11 for a description of the items in
130. annel in Offline Sorter Software may have just spikes just continuous or both spike and continuous data associated with it See Section 6 1 Details of Channels and Sources on page 277 for more information Whenever a new channel is selected in Offline Sorter Software the spike and or continuous data for that channel is automatically displayed in the Timeline view The Showing text fields at the top indicate which channels are showing in both the spike if applicable and continuous if applicable sections of the strip chart Pressing the button after the Showing text fields will open the Timeline Channels to Display dialog box In this dialog you can check uncheck the display of Spikes and or Continuous data channels for the stripchart view and select any Source and Channel for either F Timeline Channels to Display Showing Source Channel 7 Spike SPK Chs 1 64 2 SPKO2 x 7 Continuous WB Chs 65 68 66 WBO2 K oe Note that it is possible to view any spike channel along with any other continuous channel together in the Timeline view The Timeline view consists of the following components e Showing The two text fields show which Offline Sorter channel is being displayed in each section of the stripchart display Pressing the button allows you to select different channels for display e Waveform Segments stripchart display The top portion of the stripchart
131. ans on page 179 e A tool to explore different sorting scenarios by scanning the values for parameters that affect the sorting over a range of values e Section 4 5 13 Automatic Clustering Using the Scanning Methods on page 182 e The number of clusters and cluster membership is automatically determined by optimizing a cluster quality metric during a scan over values of a sorting parameter For the sorting methods that work in feature space any of the active features can be assigned to the 2 or 3 axes of feature space and used in the sorting algorithms Section 4 4 6 Selecting Features to Use For Sorting on page 143 In the examples that follow the feature space axes are generally left to the defaults values PC1 PC2 and PC3 if using the 3D Clusters view You can re sort the waveforms after the units have been selected by using the manual or automatic clustering procedures Version 4 0 145 4 Spike Sorting Step by Step 4 5 1 1 Selecting the Sorting Method This can be done either by selecting the Sort Method parameter in the Control Grid iControl Gn ME ADES Channel Data Type Single Sort Method Zoom Y Max w f draw 100000 Sort Start 0 Cart End 70 by using the Sort Change Sort Method menu command to select the method or by using the corresponding buttons on the Toolbar In general use only one of the sort methods that are described in the following sections 4 5 2 Manual Clustering us
132. applying Template Sorting 160 Applying Adaptive Template Sorting Manual Clustering using the Boxes Method Manual Clustering using the Lines Method Manual Clustering using the Bands Method Semi Automatic Clustering using K Means Selecting Initial Cluster Centroids 171 Running the K Means Clustering Algorithm Semi Automatic Clustering using Standard E M Automatic Clustering using Valley Seeking Method Automatic Clustering using T Distribution E M Method Parameter Scans 179 Automatic Clustering Using the Scanning Methods Unit Cleaning 184 Controlling Waveforms Displayed 184 Controlling waveforms viewed by Units Controlling waveforms viewed by Time Waveform Selection Options 186 Cleaning the Selection 187 Automatic Outlier Removal Tool Manual Waveform Removal 188 Combining Units 189 Swapping Units 190 Invalidating Units 190 187 Removing Spikes with Short Inter spike Intervals 191 Automatic Short ISI Spike Removal Manual Short ISI Spike Removal Rescuing Overlapping Waveforms Examining the Sorting 198 Waveform Inspection 198 192 194 Selecting Waveforms in the Timeline View Selecting Waveforms in the Clusters Views Comparing the Waveform with the Templates 200 200 Examining Sorting Statistics Info Grid Views Statistics Statistics Feedback While Sorting 203 Examining the Sorting vs Time 204 Saving the Sorting Results 205 Saving Timestamps and Waveforms 205 Overwrite Backup Control on File Saves Exporting t
133. ar or select Find Units from the right click menu If it is not cancelled the algorithm runs until convergence or until a specified maximum number of iterations has completed This Max Iterations parameter is set in the E M Sorting section of the Sort tab under the Tools Options dialog box There is a parameter in the Control Grid labelled Beta that appears when the Standard E M sorting method is selected This parameter controls the convergence properties of the algorithm for details see Section 6 5 6 Standard E M Algorithm on page 303 Version 4 0 175 4 Spike Sorting Step by Step The clustering results can be cleaned up after the sorting is completed by adding or removing waveforms from each unit e either in the Waveforms view e See Adding Waveforms to Selected Units in the Waveforms View on page 154 and e Section 4 6 3 Cleaning the Selection on page 187 e or in the Clusters views e See Adding Waveforms to Selected Units in the Clusters Views on page 150 and e See Removing Waveforms from Selected Units In the Clusters Views on page 151 4 5 10 Automatic Clustering using Valley Seeking Method Select the Valley Seeking method of sorting by selecting Valley Seek in the Sort Method drop list in the Control Grid or by selecting the Sort Change Sort Method Use Valley Seeking Sorting Method menu item The Valley Seeking algorithm uses the inter point distances in feature space to
134. are continuous recording channels within a Plexon PLX or PL2 file e Spike Waveform data It is generally desirable to record the waveforms resulting from true neural firings at higher frequencies e g 40 kHz However storing the digitized samples between the neural firings can lead to large data files Therefore neural data acquisition systems generally employ a thresholding or some other triggering mechanism to record only the digitized data in a narrow time window around what is likely to be a neural firing These waveforms segments are generally called spikes Version 4 0 9 2 System Overview e Event data Many data acquisition systems have the ability to record time stamped markers in the data stream when some external event happens While these external event markers are somewhat tangential to the software s main purpose of sorting spikes Offline Sorter Software can display them and the event markers can be used to select time periods of interest in the data Offline Sorter Software can also read in batch command files to direct its processing and files of sorting parameters that can be applied to data files For more information see Section 6 9 Batch File Processing on page 313 2 3 Sorting Techniques Basic to multi neuron study is the ability to resolve the spike waveforms associated with the multiple individual neurons which often are present on single or multiple electrodes For reviews of spi
135. are can also import data files from several other vendors and also some generic raw data files via the File Import menu selection There are several options that control how Offline Sorter Software loads data see Tools Data Import Options below Note that Offline Sorter Software can also open previously saved OMF files see File Open Multiple Files below 5 1 2 File Import Offline Sorter Software can load data files from various other vendors via these menu selections Enabling the import of neural data from these different file formats into Offline Sorter Software is a formidable task There are various limitations and factors to keep in mind for the different file formats and sometimes different vendors have different concepts and assumptions that are difficult to represent in Offline Sorter Software Import of other vendor file format into Offline Sorter Software is accomplished in one of two ways either via a native loader that was written specifically for Offline Sorter Software generally utilizing a software toolkit supplied by the other vendor or via a Neuroshare DLL see www neuroshare org As explained on the Neuroshare website the idea behind Neuroshare is for each vendor of neurophysiological data acquisition equipment to provide a Windows DLL that knows how to read and interpret the data file format created by that vendor s data acquisition system Applications such as Offline Sorter Software
136. are shipped with their available menu commands organized into a set of menus the contents of the menus can be customized and new menus can be created Icons Icons may be located immediately to the left of the menu commands These icons will be displayed on the toolbar associated with the menu In the Version 4 0 Appendix A Navigating the Plexon UI example menu there are icons associated with all but the last three of the menu commands The presence of an icon next to a menu command means that the icon is also a label on a toolbar button and will execute the same command when clicked as the menu command on the menu Hot Keys Hot Keys are key combinations that will cause a menu command to execute without having to click the command on the menu Typically hot keys will contain two keys that must be pressed simultaneously for the combination to execute The area at the far right of the menu commands may display the hot key combinations for the menu commands In the example menu there is a hot key combination CTRL C for the Copy command Hot keys may be added or customized via the Tools Customize dialog e Tear off Menu Handle The Tear off Menu Handle is an area the one containing the dots at the top of the menu present on many menus The tear off feature allows the quick creation of a toolbar that contains all of the commands in the menu that have command icons You may hover the mouse over the tool to highlight it
137. atistics e Per Unit information via the File Export Per Unit Data menu selection This item generates a line of information for each unit in the file The kind of information items that can be exported per channel include the numbers of 206 Offline Sorter Offline Spike Sorting Software Saving the Sorting Results waveforms the cluster center positions and the sigmas and other normality Statistics When exporting to a text file or to Excel you can choose an option to export a row of column header text to help in deciphering the exported data 4 8 3 1 Exporting Per Waveform Data Selecting the File Export Per Waveform Data menu selection displays the following window Which Channels ERTIES Current Channel only ane D All Channels into one file Export a Row of Column Headers All Channels one file per channel What Waveforms to Export C Export Invalidated Waveforms Z Export Unsorted Waveforms Export Only Waveforms in the Time Range From sec 0 To sec 196 733475 AlUnits OnlyUnit Unit a What to Export for Each Waveform Available Chosen Reset to Defaults Channel Name Raw Channel Name J Unit Channel Raw Add gt gt Timestamp Slice 1 PEJ Slice 2 PE Peak Add All PC3 Valley Peak Valley lt lt Remove Energy Nonlinear Energy PC 4 Remove All PC 5 PC 6 PC PC 8 MEVRA V Append Raw Waveform A D Data in units of microVolts ADC Counts
138. aveforms View by Units You can change the type of waveforms viewed in the Waveforms view by using the Waveform View parameter in the Control Grid or by using the Waveforms View Only Currently Selected Unit s menu entry To view waveforms from all units in the Waveforms view Set the Waveform View parameter in the Control Grid to All Max wf draw Sort Start Sort End Slice 1 Slice 2 ClustHist Max Outlier Thr Cluster View Ei Draw Grid Refr ISI us 1 00 ISIHistMax ms 100 Use Range E or unselect make so that it is not checked the Waveforms View Only Currently Selected Unit s menu entry 184 Offline Sorter Offline Spike Sorting Software Unit Cleaning To view waveforms from specific units in the Waveforms view Set the Waveform View parameter in the Control Grid to Sel Unit s or select make so that it is checked the Waveforms Use Only Currently Selected Unit s menu entry Select the unit by clicking on the unit s template in the Unit s Display Max wf draw 10000 Sort Start 0 Sort End 800 Slice 1 0 Slice 2 lo ClustHist Max 100000 Outlier Thr 1 View wif Cluster View i Draw Grid M Refr ISl us 1000 ISIHistMax ms 100 Note To see the waveforms that are currently unsorted select the unsorted unit in the display Note You can select multiple units as described in Section 3 4 Units View on page 42 Holding down the CTL
139. ay Features colored waveforms template average waveform for each sorted unit grey waveform waveform selected in the Timeline view or Clusters views w f number of the selected waveform in the temporal sequence of waveforms on the channel timestamp time of occurrence of the selected waveform in seconds dotted vertical lines sort start and sort stop times if present see Section 4 4 5 Adjusting the Sort Range on page 142 in upper right hand corner of each plot sum of squared difference between the selected waveform and the template Version 4 0 73 3 Views and Displays e best match displayed on the unit with the best match least sum of squared difference between the unit template and the selected waveform e Colored bar displayed on the unit assignment of the selected unit green bar above 3 11 Waveform Detection View The Waveform Detection view is only available when a channel containing continuously digitized data is opened The view displays distributions of quantities e g peak heights energy or non linear energy that can be used for thresholding the data for spike detection To display the Waveform Detection view Select the View Waveform Detection menu command or press the p button on the toolbar By default the Waveform Detection view is selected when a continuous data channel is opened The Waveform Detection view shows a histogram of the logarithm of
140. ay be an acceptable sorting so that it may be recalled if none of the what if scenarios improves the situation When saving or recalling a sorting two options are available e You can save to or recall from an arbitrary file where a user supplied file name is specified or e You can save recall from one of two pre defined slots When saving to or restoring from a pre defined slot Offline Sorter Software actually generates a temporary filename and saves to that filename But using a pre defined slot allows a single toolbar button to be pressed to save or recall a sorting which is more convenient than selecting a file with the file selection dialog To save the sorting to an arbitrary file select Sort Save Sorting to SORT File Save to File and select the file using the standard file selection dialog To restore 254 Offline Sorter Offline Spike Sorting Software Sort Menu a sorting from a previously saved file select Sort Restore Sorting from SORT File Restore from File To save the sorting to a pre defined slot select Sort Save Sorting to SORT File Save Sorting 1 or 2 or press one of the toolbar buttons iE To recall the sorting that was previously saved to a pre defined slot select Sort Restore Sorting from SORT File Restore Sorting 1 or 2 or press one of the toolbar buttons Note A SORT file is valid only for the current channel and only as long as the number of waveform
141. ays the 2D and 3D versions of each of the J3 Pseudo F Davies Bouldin and Dunn cluster validity metrics See Section 6 6 Sorting Quality Statistics on page 306 The feature space used for this sorting quality statistics calculation is displayed under Features above 86 Offline Sorter Offline Spike Sorting Software Sorting Summary View To the right of the cluster quality metrics but sharing the same vertical space is the Per Unit Statistics section which shows the L Ratio and Isolation Distances metrics for each of the units The next section displays the template average waveform for each of the units on the channel as described in the Section 3 4 Units View on page 42 The dashed line represents the 3 sigma deviation of the waveforms around the average Note The colors displayed for the units are controlled by the printing colors not the display colors The printing colors can be set in the Printing Options tab of the Tools Options dialog The bottom section is the 2D Clusters view Again the colors displayed are controlled by the printing colors not the display colors 3 14 1 Customizing the Sorting Summary The items displayed on the Sorting Summary can be customized to some degree by selecting the Customize right click menu item or pressing the associated toolbar button E Sort Summary Customization Vl Show Filename in Title V Show Extraction Information for continuous data
142. be supported in the future Tolerance 70 Template Sort Units Minimum Overlap Run Overlaps Analysis Unit Permutations 6 Overlap Templates 45 Matched Overlaps 89 Showing Units a b lt Prev Next gt Show on Clusters Views v Show Unit Shift Text Rescue Overlaps The method used for Overlaps Analysis is a somewhat brute force matching algorithm Each permutation of existing unit templates is considered separately The analysis results for a single pair of unit templates at a time is shown on the Overlaps Analysis screen the results for the other unit templates pairs can be inspected by pressing the Next gt and lt Prev buttons to scroll through them The total number of unit permutations is shown on the left under Unit Permutations For each pair of unit templates e g Unit a and Unit b in the above screenshot the analysis works by superimposing offset versions of the two unit templates then determining where the superimposed signal would have crossed the detection threshold then using the Waveform Width and Prethreshold settings to extract a Overlap Template waveform from the superimposed signal The Overlap Templates for different offsets are shown in purple in the Overlaps Analysis grid shown above The number of samples that each unit template was offset the Unit Shifts is shown in the light blue text in a format where the shift of the first unit template is the first number fol
143. ber of tables for the p values and each of the Sorting Quality Statistics which are detailed on page 306 for both 2D and 3D feature spaces Each table has the units listed across both the rows and the columns the value at the intersection of the ith row and the jth column represents the pairwise statistic calculated for Unit i and Unit j The value is calculated as if only those two units existed in the channel and only the waveforms assigned to those units existed All other waveforms in other units or unsorted are ignored Note In each table the matrix formed by the values is symmetric a i j aG i and the diagonals that correspond to the pairwise statistics calculated between some unit and itself are meaningless and are set to 0 The values in the tables in some sense indicate how well sorted each pair of units is A poor sort quality value between two units can indicate that the two units need to be combined into a single unit Take care with the interpretation of these pairwise results as they are a post hoc test and the probability is high that somewhere in the tables there are values that indicate exceptionally well sorted units The calculation of these values can take a noticeable amount of time to complete Because of this the calculation is optional and it is disabled by default To enable it check the Calculate unit pairwise statistics after each change checkbox in the Tools Options Stats dialog box
144. between the two windows The illustration below shows the Marker Occurrences window without sidebar tabs on the left On the right a Channels window has been placed into the same space by using the center docking sticker Note that there are two sidebar tabs at the bottom of the window A 8 Offline Sorter Offline Spike Sorting Software sig001 Spikes sig002 Spikes sig003 Spikes sig004 Spikes sig005 Spikes sig006 Spikes sig007 Spikes sig008 Spikes sig009 Spikes sig010 Spikes sig011 Spikes sig012 Spikes sig013 Spikes sig014 Spikes sig015 Spikes sig016 Spikes sig017 Spikes sig018 Spikes sig019 Spikes sig020 Spikes sig021 Spikes sig022 Spikes sig023 Spikes sig024 Spikes sig025 Spikes sig026 Spikes sig027 Spikes sig028 Spikes sig029 Spikes sig030 Spikes sig031 Spikes sig032 Spikes 4DO1 Continuous 4DO2 Continuous ADOS Continuous 4D04 Continuous DOOOOOO OOOO OOOO OOOO See oan ww pe RE co co e Hidden Windows Docked windows that you do not need to access often can be hidden or rolled up by pressing the Auto Hide button When hidden the window is represented by a sidebar tab Sidebar tabs may be located at the left right or bottom of the screen and indicate hidden windows To show one of these windows just hover the mouse over one of the tabs and the window will appear The File Info window of CinePlex Editor opened by the sidebar tab is shown below on the left If you click the Auto Hide contro
145. bination will be the key assignment for the selected command 6 Click the Assign button to assign the key combination to the selected command If there already is a key assignment for the command a confirmation box will display to allow you to confirm or cancel the reassignment Note In CinePlex Editor do not bind menu operations to keystrokes that are associated with markers Use only CTRL or ALT key combinations to bind to menu items as these key combinations cannot be associ ated with markers A 4 4 Run Menu Customization The Run Menu tab allows you to manage menu items on the Run Menu An illustration of the Run Menu tab follows Customize Toolbars Commands Keyboard Run Menu Options Menu contents Offline Sorter NeuroExplorer Explorer Command PlexUtil exe Arguments Initial Directory e Menu contents This is a list of all the menu items in the Run Menu Select an item to see its characteristics in the Command Arguments and Initial Directory areas Version 4 0 A 21 Appendix A Navigating the Plexon UI Buttons The toolbar buttons allow you to add a new menu item delete a selected menu item move the selected menu item upward in the list and move the selected menu item downward in the list Command This area contains the name of the file that rans when the menu item is selected from the Run Menu The browse button opens the standard Open dialog box filt
146. ble of using an existing set of sorted units as a starting point in what is referred to as Continue sorting The algorithms can run using the centroids or template waveforms of existing clusters as the starting points instead of manually specifying them Automatic sorting methods The Automatic sorting methods are capable of automatically finding the optimal number of clusters and of assigning waveforms to the clusters Thus they require Offline Sorter Offline Spike Sorting Software Sorting Techniques no user intervention to arrive at the initial clustering of the data and they can operate on completely unsorted data The Valley Seeking automatic method uses a Valley Seeking algorithm that it applies to inter point distances to automatically determine the number of clusters and the cluster memberships The T Distribution E M T Dist E M method is another variation of the Expectation Maximization algorithm that fits a mixture of T Distributions instead of Gaussians to the point densities in feature space This algorithm is also capable of adjusting the number of clusters as it runs by removing unfavorable clusters to arrive at the optimum number of clusters The Scanning methods are a family of automatic sorting methods that attempt to find an optimal clustering by stepping a sorting parameter a value that controls how the sorting is performed through a range of values One of the Sorting Quality Statistics describe
147. both 2D and 3D The numerical values are in units appropriate to the corresponding feature in the current feature space You can also export the features selected for each axis of feature space as a numerical code In addition to or instead of the exported feature values Offline Sorter Software can additionally export the raw A D values associated with each waveform To do so click the Append Raw Waveform A D Data checkbox The A D values appear in additional data columns after the exported feature columns To export only the waveform A D values deselect all features from the Chosen list box and click the Append Raw Waveform A D Data checkbox The number of appended columns is equal to the number of points per waveform The waveform A D values can be exported in units of millivolts the default or in raw ADC counts by clicking the radio buttons near the checkbox Select What File to Store the Exported Data in by typing a filename or selecting one using the Browse button To append the data onto the end of an existing file click Append if existing Click Export For exports to Excel save the spreadsheet using Microsoft Excel 4 8 4 Saving Template Files Template files can be used to apply the sorting done on one channel to a different channel A common usage of this is to apply the same sorting done for one file channel for channel to another file that was acquired with the same experimental setup For this case consider using th
148. button on the toolbar The Clusters vs Time view shows the waveform points in 2D feature space but time is plotted as the third axis with the start of the file at the bottom and the end of the file at the top of the screen initially Thus a perfectly stable unit whose spikes occurred continuously during the experiment will show up as a continuous vertical column of waveform points The Cluster vs Time view behaves identically to the 3D Clusters view as for how the view point can be rotated and zoomed See Section 3 7 2 13 3D Clusters View Details on page 65 Also all of the functionality to create and edit units using Contours is available in the Clusters vs Time view 72 Offline Sorter Offline Spike Sorting Software Waveform Inspection View 3 10 Waveform Inspection View The Waveform Inspection view is used for comparing individual waveforms with the template average waveform for each sorted unit To display the Waveform Inspection View Select the View Waveform Inspection menu command or aa click on the toolbar The Waveform Inspection Window shows the average waveform template of each sorted unit in the appropriate unit color The individual waveforms which are selected either in the Timeline View left click on waveform or in either of the 2D and 3D Clusters Views left click on waveform s dot in feature space are displayed in grey and positioned over each template for comparison Displ
149. can be active at the same time 52 Offline Sorter Offline Spike Sorting Software 2D and 3D Clusters Views 3 7 1 Displaying 2D and 3D Clusters Views To display the 2D Clusters View Click the 2D Clusters toolbar button or select the View 2D Clusters menu command ps S x g amp Add Unit Find Units Delete Unit Add Wfs Remove Wfs Invalidate Wfs Version 4 0 53 3 Views and Displays To display the 3D Clusters View 3D Press 3D Clusters toolbar button command or select the View 3D Clusters menu Features common to both the 2D and 3D views are discussed in the Clusters Views Common Functionality section that follows Features specific to either view are discussed in Section 3 7 2 12 2D Clusters View Details on page 61 and in Section 3 7 2 13 3D Clusters View Details on page 65 3 7 2 Clusters Views Common Functionality The following features are common to both the 2D and 3D Clusters views 3 7 2 1 Axis Feature Selection The variables shown on the X Axis and Y Axis for the 2D Clusters view and the Z Axis for the 3D Clusters view can be selected from among any of the Section 6 4 Features Available for Sorting on page 294 by using the X Axis Y Axis and Z Axis Drop Down Feature Lists on the Toolbar Only the currently active features show up in the drop down feature lists The currently active features in Offline Sorter Software are a subset of the Sectio
150. cel spreadsheet The information to be written and other controlling parameters must be specified in the File Export Per Channel Data dialog 6 9 1 4 ForEachChannel The ForEachChannel command specifies an action to be done on each channel of each file Syntax ForEachChannel sub command e g ForEachChannel ValleySeek Sub commands for the ForEachChannel command are e Upsample performs interpolation to upsample each channel of continuous data as configured with the Set UpSampleXXX commands e Filter applies a low cut filter to each channel of continuous data The filter cutoff frequency is specified with the Set FilterFreq command Version 4 0 315 6 Reference Topics Detect performs waveform detection for each continuous channel as configured with the Set DetectXXX commands Align runs waveform alignment using the currently set alignment parameters set via the Set AlignXXX commands DeleteAllUnits removes all existing units Sort performs sorting that has been specified via the Set SortType command ValleySeek ValleySeek2d runs Valley Seeking sorting on each channel using 2D feature space ValleySeek3d runs Valley Seeking sorting on each channel using 3D feature space TDist TDist2d runs T Distribution E M sorting on each channel using 2D feature space TDist3d runs T Distribution E M sorting on each channel using 3D feature space RemoveOutliers2d RemoveOutlier3d
151. cian ea emt rere 109 4 2 Waveform Acquisition From Continuously Recorded Data 0 00 110 4 2 1 Opening the Digitized Data Files cceeseeeeeseeeeeseeeeeeeeestteeeeeees 110 4 2 2 Selecting the Continuous Data Channel ccccscesseeeeesesneeeees 111 42 8 Speciiyng U PHS ai riro aaa 114 iia MTs T E Uy e ere E E meen T sere A rer E EE trr arn 115 4 2 5 Selecting a Digital Filter Low cut or High Cut csceeseeeees 118 4 2 6 Specifying the Waveform Detection Options ccccceeeeeeees 120 4 2 7 Extracting the Thresholded Waveforms csseeeesessteeeeeeenaaes 123 4 3 Waveform Acquisition From Spike Waveform Data cccccceeeeeee 124 4 3 1 Opening the Spike Waveform Data File cccccsecceesseeeeesseneeees 124 4 3 2 Stereotrode and Tetrode Data Type Selection ccceeeeeeeee 125 4 3 3 Selecting NS Data Channel soosicceculsccinsuwaiseassivnsuetaanosstaciuisesisans 125 AA Preparation For Sorring seisoon eenain i i 127 AAA Data NepociON scia 127 442 invalidating VF AV STAINS ensnare 129 AS Examining Evos sanna 137 4 4 4 Wavelonm Alignment cissccccoccussensccnrsunmenccnrmmenaecstinmeccsmmumnonnsmmwences 138 4 4 5 Adjusting the Sort Range senna aaa 142 4 4 6 Selecting Features to Use For Sorting cccccccssseeesseeeesteeeeees 143 Rotae g a 119 5 E an peur ee rere eer E bo A reer re et prer Pr arene eer reer tere 143 4 5 1 Manual Semi Automatic and
152. d and individually colored and toggled on off and can be saved and restored as NeuroExplorer intervals See Section 3 2 5 Time Segments View on page 34 Additional information was added regarding the stripchart display in Timeline view which can show both spike and continuous data for a single channel See Section 3 6 1 Stripchart Display in the Timeline View on page 48 Information was added regarding the algorithm the software uses to calculate the noise sigma used in the Waveform Detection dialog See Section 3 11 1 Threshold Calculation on page 75 The Show Tetrode Min Max Electrode Combinations option was added to the Feature vs Feature view See Section 3 13 Feature vs Feature View on page 80 A Sort Summary Customization option was added in the Sorting Summary view See Section 3 14 Sorting Summary View on page 84 The ability to apply high cut filtering to continuous channels was added See Section 4 2 5 Selecting a Digital Filter Low cut or High cut on page 118 Bands and Lines methods of manual clustering were added See Section 4 5 1 Manual Semi Automatic and Automatic Clustering Methods on page 143 Scanning methods were added in the Parameter Scan grid view This includes the ability to scan using different random initial seed clusters See Section 4 5 12 Parameter Scans on page 179 A procedure was added for rescuing overlapping waveforms See Section 3 22 Overlaps Ana
153. d bottom of the Clusters views can be on following left or off following right ok i Ad i x po 2 2 id Unit Find Units Delete Unit Add Wfs Remove Wfs Invalidate Wfs E p 2 01917e 031 J3 11 PsF 11114 8 DB 0 309 Dn 1 62 To show or hide the toolbar click on the triangle in the windows title bar or right click in the title bar and select the Toggle Toolbar menu option The text box at the top of the display shows the current value of a selected cluster quality metric along with the percentage change of that metric that resulted from the last sorting related change If the last change improved the cluster quality the box appears in green and if it degraded the cluster quality the box appears in red This can be very useful when deciding which unit to assign borderline waveforms to Any of the available cluster quality metrics described in Section 6 6 Sorting Quality Statistics on page 306 can be used for this display the metric can be chosen in the Stats tab of the Tools Options dialog box To show or hide this text box select the Show Cluster Quality menu option in the right click menu The text box at the bottom of the screen will either show the values of all Sort Quality Metrics described in Section 6 6 Sorting Quality Statistics on page 306 above or an operational hint below depending on context 58 Offline Sorter Offline Spike Sorting Software 2D and 3D Clusters Views c Fhe Z
154. d for performing remote upgrades to unlock additional features or product licenses Use the following procedure to check Plexon licenses Version 4 0 1 Getting Started Note To open the license management window from the main menu select Help About Offline Sorter then press the Licensing button to bring up the Plexon License Management window r PlexLic Tool v4 0 0 Mar 29 2015 Sentinel Versions Library 7 1 Driver 7 5 0 or later Key fue FLeCXON Key Serial Number 03225 Neurotechnology Research Systems Original Customer ID 00866 Key Number for Original Customer O0006 Products Licensed Offline Sorter YES 2 x 3 x and 4 x WaveTracker YES Recorder NI NO Recorder ADS NO Rasputin NO NeuroSurgery WS NO MEA WS NO PL2 NO CinePlex NO CPN 3 NO CineLab V4 NO OmniPles NO Radiant NO Test the Key Again Unlock Additional Programs and Features i 2 3 4 Enter Code In the main text area the window contains a summary of the licensing status of the licensing key or keys If Offline Sorter Software cannot read a licensing key an explanation appears in the window If Offline Sorter Software can read a licensing key it interrogates it and displays the information from it The Key Serial Number Original Customer ID and Key Number for Original Customer identify a particular key This information must be provided to Plexon to unlock additional Plexon pr
155. d in Section 6 6 Sorting Quality Statistics on page 306 is chosen as the metric that defines what optimal means For example Valley Seeking automatic sorting method has a sorting parameter called the Parzen Multiplier that affects how the clustering proceeds The Valley Seeking Scan sorting method will step the value of the Parzen Multiplier through a user defined range of values and calculate the Sorting Quality Statistics for each step The step that produces the best value of the chosen Sorting Quality Statistic is taken as the final sorting Working with sorted data inspecting adjusting and resorting Offline Sorter Software provides many mechanisms for inspecting and manually adjusting the sorting results it is possible to add or remove waveforms from clusters or invalidate them completely With Offline Sorter Software the waveforms can be resorted by using any of the clustering methods After the units have been defined the contours templates and principal components can be saved to a TPL file and they can be used to sort waveforms in other data files For more information on the sorting algorithms see Section 6 5 Details of the Sorting Algorithms on page 299 References Lewicki M S A review of methods for spike sorting the detection and classification of neural action potentials Network Comput Neural Syst 9 R53 R78 1998 Schmidt E M Computer separation of multi unit neuroelectric data a review
156. d the Timeline view when the Energy of the Signal threshold has been selected l RT b Hi Ui i IF 4 2D Clusters 3D 3D Clusters Timeline Control Displays Center Selected Zoom 1 0 5 00 mY Thr 1225 12 22 m Detect Waveforms Options Up Sampling None z Frequency Low cut Filter None X Scroll Range 10 0000 0 0000 Zoom Qut Zoomin lt The top histogram in the Waveform Detection view above shows the distribution of the logarithm of the signal energy and the top trace in the Timeline view shows the waveform energy The thresholding is set in the top histogram or trace Offline Sorter Software calculates the Energy of the Signal by taking a moving window average of the square of the voltage amplitude The width of the window used for the averaging is controlled in the Detector and Waveform Options dialog Nonlinear Energy is akin to the product of the raw amplitude times the derivative of the signal If the voltage is constant or zero the Nonlinear Energy is zero If the waveform is rapidly varying and has a large amplitude the Nonlinear Energy is a maximum Version 4 0 77 3 Views and Displays Mathematically the energy and nonlinear energy of the signal at time point i is given by 1 Bnergy i vi vO Nonlinear Energy i ZO va v vd 0 where is the sum over the energy window centered around i i e the sum over j w
157. d units 1 Select the unit click the unit s template display 2 Select Units Add Waveforms to the Selected Unit menu command 3 Move the mouse pointer to the Clusters view eet The pointer changes to a blue drawing hand for adding waveforms 4 Click the left mouse button and drag a line around the units to be added 4 6 5 Rescuing Overlapping Waveforms This section provides guidance on using the Overlaps Analysis feature For a discussion of the basic principles and display of overlapping spikes see Section 3 22 Overlaps Analysis View on page 99 The use of the Overlaps Analysis feature in Offline Sorter Software rests on a number of assumptions and therefore the best results will be achieved if a specific workflow is followed 1 Detect Spikes with Dead Time at least as long as the Waveform Length Overlaps Analysis can only be used if spikes were extracted within Offline Sorter Software using waveform detection Overlaps Analysis cannot be used on spikes that were recorded during the experiment 194 Offline Sorter Offline Spike Sorting Software Unit Cleaning When doing the waveform detection use only a single threshold on the simple Raw Signal detector And make sure that the Dead Time specified in the Waveform Detection Options dialog see Section 4 2 6 Specifying the Waveform Detection Options on page 120 is at least as long as the Waveform Length r Waveform Detection Options Extracted Wa
158. daptive Template Sorting has been performed It shows the template waveforms plotted against time which shows how the waveforms changed as a function of time See the discussion on page 163 in Section 4 5 4 3 Applying Adaptive Template Sorting Version 4 0 23 3 Views and Displays Waveforms Inspection View The Waveform Inspection view compares individual waveforms selected in the Timeline view with the average waveform template of each sorted unit For more information see Section 3 10 Waveform Inspection View on page 73 Waveform Detection View The Waveform Detection view is only available for channels with continuously recorded signals It displays histograms of detected peak heights that allows a spike detection threshold to be set For more information see Section 3 11 Waveform Detection View on page 74 Continuous Frequency Spectrum View The Continuous Frequency Spectrum view is only relevant for channels that contain continuous data samples It displays the results of a Fast Fourier Transform performed on a span of continuous data samples showing the amount of energy at each frequency See Section 3 12 Continuous Frequency Spectrum View on page 80 Feature vs Feature View The Feature vs Feature view displays miniature 2D Clusters view windows for various combinations of features It is possible to identify features quickly that are the most useful in the sorting of units For more information see
159. ded from the Plexon website http www plexon com 2 Select File Execute Batch Command File navigate to the SampleBatchFiles directory located at C Program Files Plexon Inc Offline Sorter 3 SampleBatchFiles for a default installation and select the desired sample batch command file 3 After the batch command file runs inspect the results for example by selecting File View Log from Last Executed Batch Command File The sample files are 6 10 1 SortAndSave ofb Performs Valley Seeking sorting using 2D feature space on all channels in a file performs outlier removal then saves the results Note that this batch command file must be run on a data file that contains spikes and it must be run on a type of data file that Offline Sorter Software can save natively i e the File Save and File Save As menu items are available after loading the file After the batch command files completes the output should be in a file with the same name as 324 Offline Sorter Offline Spike Sorting Software Large File Tips the currently open file with a number appended e g if the current file is test plx the output file will be test 01 plx There should also be a SortAndSave ofb log file in the same directory as SortAndSave ofb that records what happened during the batch command file execution 6 10 2 RemArtifactsSortAndExport ofb Performs artifact invalidation followed by T Distribution E M sorting using 3D feature space
160. degree of separability even though the amplitude variance may not be large are emphasized in the projection In many cases the net result of Enhanced PCA is to make the feature space clusters more compact and distinct However the degree of improvement is data dependent and so you should compare standard PCA and Enhanced PCA to determine which is optimal for your application Here is an example of standard PCA top versus Enhanced PCA bottom 312 Offline Sorter Offline Spike Sorting Software Batch File Processing 6 9 Batch File Processing When a command file executes Offline Sorter Software shows the progress in a dialog and also writes a log file with a log extension appended to the original batch command file name Offline Sorter Software also has a convenience command File View Log from Last Executed Batch Command File that will quickly show the log file from the last batch execution in the current editor Offline Sorter Software will recognize a command line argument b BatchFileName so that a batch command script can be executed from the command line or from a Windows bat file For example OfflineSorter b test ofb at the command prompt will start Offline Sorter Software immediately execute the test ofb batch command file and then close Offline Sorter Software 6 9 1 Batch File Commands The format of the batch command files is one command per line A command consists of a main keyword followed
161. dition to the originally present units The final step in the sorting process is to remove any units that are empty units with no waveforms in them 6 6 Sorting Quality Statistics There is no universally agreed upon way to measure sorting quality so Offline Sorter Software can calculate and display a number of different sort quality metrics It can also calculate pair wise cluster separation statistics and also other supporting statistical data such as measures of normality of distributions The following sections describe the sort quality metrics and other statistics available in Offline Sorter Software You can view these values on the Sorting Summary View which is detailed on page 84 or on the Stats view of the Info Grid Views which is detailed on page 25 Also you can choose one of the sorting quality metrics to be displayed on the top of the 2D and 3D Clusters Views which are detailed on page 52 where it can be used to give immediate feedback on the effectiveness of sorting decisions See Statistics Feedback While Sorting on page 203 306 Offline Sorter Offline Spike Sorting Software Sorting Quality Statistics 6 6 1 MANOVA F and p values The degree to which the selected unit clusters are separated in the 2D and 3D Clusters views is determined by a Multivariate Analysis of Variance MANOVA test The Null hypothesis in the MANOVA is that all clusters actually come from the same underlying statistical distrib
162. dsans aae a Aae Dear aa RR AS 273 STS OWCE Beale We NICS eiar a ANa 274 w LA AGO ONIME SOE e inaa i ENa AAAA REANA 274 Version 4 0 219 5 Menu Options 5 1 File Menu The File menu contains operations such as opening saving and exporting and closing data files executing batch command files and printing sorting summaries Open Ctrl O Import gt Open Multiple Files Close Save Ctrl S Save As Export to New PLX Export to New PL2 Export to NEX Export Per Waveform Data Export Per Unit Data Export Per Channel Data Execute Batch Command File Edit Batch Command File View Log from Last Executed Batch Command File Print Ctrl P Print Preview Print Setup Print All Sort Summaries Send All Sort Summaries to PowerPoint 1 Tetrode all data 64 channels plx 2 Tetrode all data 64 channels pl2 3 Tetrode all data 64 channels nex 4 Tetrode all data 64 channels pl2 5 Stereotrode all data 64 channels nex 6 Stereotrode all data 64 channels plx 7 Stereotrode all data 64 channels pl2 8 Stereotrode all data 64 channels pl2 9 64 channels alldata units nex 10 64 channels alldata units plx Exit 220 Offline Sorter Offline Spike Sorting Software File Menu 5 1 1 File Open The standard document types for Offline Sorter Software are the Plexon data file formats PLX and PL2 These files are opened from the File Open menu Offline Sorter Softw
163. e If unchecked the default all Continuous data and Events will be exported even if a Time Range is specified e Range of Samples within Waveforms to Export The item enables short ened waveforms to be stored in the output file For example if the sort range is reduced only the relevant portion of each waveform can be saved If All Samples the default is selected the entire waveform is saved If Only the Samples is selected only the samples in the specified range are written out for each waveform 5 1 8 File Export to New PL2 If Offline Sorter Software has loaded a PL2 file it can also write out a new PL2 file The Export to New PL2 option must be used to save PL2 files if waveform extraction alignment or any other operation that prevents File Save from being available has been used Select the File Export to New PL2 menu command to bring up the following dialog box 232 Offline Sorter Offline Spike Sorting Software File Menu r Export to New PL2 Options C PlexonD ata pl2 test files all data 64 channels 01 pl2 Sources to Export Id Source Chs Freq 7 SPK 1 64 40000 vj 4 WB 65 128 40000 iv 5 SPKC 129 192 40000 jv 8 FP 493 256 1000 v 9 KBD 340 F0 10 Single bit events naia To yl 11 Other events 43 45 0 f 12 CinePlex Data 46 48 0 1 I SPK_SPKC 129 192 40000 Export Invalidated Waveforms 7 Export Unsorted Waveforms Cance
164. e Adaptive Templates History view is only relevant if an adaptive templates sort has been performed It shows all the templates used for each unit with the time that each template was put into effect This is a tabular view of the same information that is displayed graphically in the Templates vs Time view See Section 4 5 4 3 Applying Adaptive Template Sorting on page 160 Note that the templates will not be displayed if Offline Sorter Software is running in Demo mode 3 2 13 Channel Map View The Channel Map view shows how the raw channel numbers from the data files map to the Offline Sorter channel numbers Each Offline Sorter channel has a line that shows which raw channel s are associated with it For more information see Section 6 2 Channel Mapping on page 286 The Channel Map view can be used to change the Data Type and to map and re map raw channels in arbitrary ways 3 2 14 Messages View When Offline Sorter Software does some significant processing or otherwise reaches a milestone it makes an entry into a Message Log to that effect This Messages view displays the contents of the Messages Log Consulting the Messages view is useful if it becomes necessary to debug a problem with Plexon Support and also as a reminder of what actions were taken recently The right click menu in the Messages view contains convenient entries to select and copy the log to the Windows clipboard to save the log to a text file and to prepare
165. e Control Displays check box and the Waveform View parameter in the Control Grid can be used to control which waveforms are displayed in the Waveform View If you limit the waveforms displayed in this way it can often reveal structure that is hidden when all waveforms are drawn By default Offline Sorter Software automatically determines the zoom factor for the Waveform View and the Timeline View The zoom factor can be disabled by unchecking the Auto Zoom checkbox in the Tools Options Display dialog box The auto zoom calculation finds the peak amplitude present in any spike Version 4 0 127 4 Spike Sorting Step by Step waveform and sets the zoom to the largest integer value that displays the waveforms without clipping them The Auto zoom procedure can be performed at any time by selecting the Waveforms Auto Zoom Waveform Displays menu item Offline Sorter Software reads spike waveforms from a file or detects them from continuous data and stores them in memory You can invert the waveforms stored in memory by using Tools Invert Waveforms on the main menu After an Invert Waveforms operation is done all principal component and feature calculations use the inverted version of the waveform If Export to New PLX or Export to New PL2 is selected Offline Sorter Software writes the inverted version of the waveforms to the output file However if a normal Save or Save As operation is done Offline Sorter Software does
166. e Fit Tolerances after Each Change Calculate Fit Tolerances from Existing Units 5 7 1 Units Select Unit s Shows a simple dialog that allows selecting the current units Version 4 0 257 5 Menu Options Select Multiple Units Unsorted Unit a Unit b Unit d Unit e This is equivalent to selecting units in the Units view as described in Section Selecting Units on page 43 5 7 2 Units Delete Selected Unit Deletes the selected unit all waveforms previously sorted into that unit are set to unsorted Any other sorted units higher than the removed unit are shifted down 5 7 3 Units Delete All Units Removes all unit definitions and returns all waveforms to Unsorted 5 7 4 Units Combine Units Shows the following dialog 258 Offline Sorter Offline Spike Sorting Software Units Menu Combine Units Select the units that will be combined into a single unit by clicking on the units in the list box Eee After selecting two or more unit designations and pressing OK Offline Sorter Software will combine all the selected units into a single unit with the lowest unit designation That is in the above screenshot all waveforms in Units a c and d will be put into Unit a 5 7 5 Units Swap Units Shows the following dialog Swap Units Select two units that will be swapped by clicking on the units in the list box Unit e Version 4 0 259 5 Men
167. e Load Multiple Files feature of Offline Sorter Software This feature allows multiple PL2 files only to be loaded and sorted together resulting in the channels in all files being sorted in the same way 4 8 4 1 Saving Templates As discussed above in the Spike Sorting Step by Step chapter after selecting units by using the manual or automatic clustering methods a template can be used to resort the spikes You can save the following items in a TPL text file templates 212 Offline Sorter Offline Spike Sorting Software Saving the Sorting Results e contours e tolerances e sort widths e feature selections e principal components e thresholds filters and detection parameters for continuous data files You can then use the saved information to perform sorting on other data files or export it to other analysis programs Sorting using saved templates is useful for files recorded in succession with the same number of channels gain on each channel and global sorting parameters waveform length and prethreshold time If Contour sorting is used to sort a channel Offline Sorter Software saves the contour information in the TPL file It also saves the sort method used for the channel in the TPL file so some channels can be resorted using templates and some with contours If waveform extraction has been performed on a channel with continuous data Offline Sorter Software saves the extraction parameters in the TPL file
168. e Principal Components Recalculate Principal Components Using Use Enhanced PCA Waveforms To Use All the waveforms All valid unsorted waveforms Use only waveforms within the time range Maximum waveforms to use 170000 Waveform Points To Use All the points Only the points between sort start and sort end Do not show this dialog again Checking the Use Enhanced PCA checkbox will perform a variant of the Principal Components calculation described in Section 6 8 Enhanced PCA Feature Space on page 312 You can restrict the PCA Calculation to use only a certain number of waveforms This is a useful feature for larger files because the PCA calculation for channels containing large numbers of waveforms can be time consuming All of the options that are available in the Recalculate Principal Components window are also available under the Feature Calc tab of the Tools Options window Offline Sorter Software remembers all of the options that control the PCA calculation between invocations To control which portion of each waveform is used in the Principal Component calculation under Waveform Points to Use click either the All the points option to use the entire waveform or the Only the point between sort start and sort end option to use only the subset of points between sort start and end Click Do not show this dialog again to suppress the display of the Principal Components window after selec
169. e Sorter Offline Spike Sorting Software Plexon Inc Chapter 6 Reference Topics 6 1 Details of Channels and SOUS caiiscccctsec trees cvastiareanctbntnaceeunieceietinliaadseaiad 277 EI Rav Channels and SOUTE E nronadiai n N aN petits 277 6 1 2 Offline Sorter Channelen iain a aa 278 6 1 3 Offline Sorter Channels and SOurces ccc ccsssscccecsccosssncesscccesseereassencceniacteaeecs 278 6 1 4 Viewing Corresponding Channie Sisca 282 Be ree MAUI arara AN ENA E Aa 286 6 2 1 Stereotrode and Tetrode Channel Mapping c ccccseseeeseeeeeeeeteeeeeeeetees 288 Gae Convertino Data TPE enia a ea i ai 288 6 2 3 Loading a Channel Map from a FING sitciccerssceessiite pac racntsianicad wankeniaadae sls 290 6 2 4 Saving a Channel Map to amp File costs csisnsecasssestcieeteenesta cusses antabe teases 291 6 3 Steregirode and Tetrode Dala suenen aaiae Aa 291 6 3 2 Extracting Stereotrode or Tetrode Spikes from Continuous Data 0 292 6 3 3 Sorting Stereotrode and Tetrode Spike Waveforms cccccseeeeeteeesteeeees 293 SA Disapa TERS hie Sa cs aacascwbdnledinedeadaledacmnalediandoes ik deemeammeindecss 294 GA Features Available for SOING cccctescs tyre Sade ip snc aaia ii 294 DAI Punpa compone S aiiai a 297 6 4 2 Voltage at Specific Slices in Time ssiiriniisniiniiiseinonineona aiiin 299 6 5 Details of the Soning AU GOM NIS sas sicsizcess ancsnesentacetscassesaddopesenesacanctmnvadeateauarentnesens 299 6 5 1 Feature Space
170. e Sorter Software does not write the invalidated waveforms to the file e Export Unsorted Waveforms If this item is selected the default Offline Sorter Software writes out the waveforms that are not sorted If unselected Offline Sorter Software writes out only sorted waveforms e Export Continuous Data Select this item to store continuous data channels in the PLX file If this item is not selected continuous channels are not stored in the PLX file even if there are continuous channels in the current data Export Up Sampled Filtered Continuous Data Select this item to store the up sampled if relevant and low cut filtered if relevant version of the continuous data If this item is not selected the raw unprocessed continuous data is stored in the PLX file e Export Only the Selected Continuous Sources If checked only the continuous Sources that are checked in the table are exported to the output PLX file Time Range to Export If Entire File is unselected the default Offline Sorter Software writes all waveforms out regardless of their timestamps If Only Data from within the Time Range is selected Offline Sorter Software does not write waveforms with timestamps outside the specified range to the output file A Time Segment can also be used to define the Time Range The Time Range Limit Also Affects checkboxes control whether the Continuous data and Events will also be limited to the specified Time Rang
171. e Wfs Invalidate Wfs Delete Unit Add Wfs Re Move the mouse pointer to the Waveforms view sy The pointer changes to a drawing hand Version 4 0 153 4 Spike Sorting Step by Step 2 Click the left mouse button and drag a line across the bundle of waveforms Note Up to 26 units can be sorted for each data channel 4 5 3 2 Adjusting the Sorting Adding Waveforms to Selected Units in the Waveforms View Waveforms can be added to existing units by crossing waveforms in the Waveforms view 154 Offline Sorter Offline Spike Sorting Software Spike Sorting To Add Waveforms to a unit green unit above 1 Select the unit by clicking it s template in the Units view amp amp I or Select the Units Add Waveforms to the Selected Unit menu command 3 Move the mouse pointer into the Waveforms view lt The pointer changes to a drawing hand 4 Click the left mouse button and drag a line across the waveforms to add The crossed waveforms are added to selected unit Version 4 0 155 4 Spike Sorting Step by Step Removing Waveforms from Selected Units in the Waveforms View Waveforms can be trimmed away from existing units To Remove Waveforms from a Unit 1 Select the unit by clicking it s template in the Units view re i amp Add Unit Delete Unit Add W Remove Wfs invalidate Wfs E or select the Un
172. e button and drag a line across the artifact waveforms The invalidated waveforms appear as dashed lines 2500 my 1000 usec Version 4 0 131 4 Spike Sorting Step by Step The invalid waveforms are ineligible for unit selection in any of the spike sorting methods You can remove them from the Waveforms view and the Clusters view by setting the Invalid w f parameter in the Control Grid to Hide pe ee aay eo Sa Draw Grid You can undo the invalidation operation by using the undo mechanism or by selecting the Waveforms Mark All Waveforms As Valid menu command 4 4 2 2 Marking Noise Waveforms as Invalid Using Clusters Views Artifact waveforms generally appear as outlying points on the Clusters views You can mark the points as invalid by using a procedure similar to that described in this section Another reason to mark waveforms as invalid is because they are due to noise that does not represent a valid neural action potential This section describes how to invalidate noise waveforms when the 2D Clusters view is showing PC1 vs PC2 You can use the same procedure in the 3D Clusters view and in any feature space The principal components of the extracted waveforms represent linear combinations linearly independent projections of the waveforms which account for maximal variance in the waveform data Maximal variance implies maximal separation of the noise cluster from the unit clusters and the unit cluster
173. e channel where rarely firing units perhaps are not completely washed out by the frequently firing units 268 Offline Sorter Offline Spike Sorting Software Tools Menu The Density Leveling tool can be run multiple times each time it follows the same recipe Running it a large number of times will eventually result in almost all waveforms being invalidated 5 8 13 Tools All Options Offline Sorter Software has a large number of options or settings that control its behavior Menu items here allow resetting these options to the factory defaults via the Tools All Options Reset to Defaults menu item and also saving and restoring all the settings to a file using Tools All Options Save to File and Tools All Options Restore from File Note that it is very good practice to save the options to a file and then specifying that options file in the Set OptionsFromFile Offline Sorter batch command This will make sure that all options are set to good values for the batch command run Version 4 0 269 5 Menu Options 5 8 14 Tools Options Offline Sorter Software has a large number of options or settings that control its behavior Selecting this menu item or pressing the toolbar button will launch the Options dialog l os Use Only Unsorted Waveforms Remove Outliers Tool Remove Outliers from All Units Only the Currently Selected Unit s Save and Restore
174. e cluster Note In the 3D Clusters view contours are drawn in screen space the projection of the 3D feature space points onto the 2D screen So the Contour sorting operation is performed on a 2D projection of the 3D feature space Up to 26 units can be sorted for each data channel 4 5 2 2 Adjusting the Sorting After any sorting not just sorting performed using the Contours method Offline Sorter Software can be used to adjust the sorting by adding or removing individual or groups of waveforms from clusters or deleting units entirely Version 4 0 149 4 Spike Sorting Step by Step Adding Waveforms to Selected Units in the Clusters ViewsWaveforms can be added to existing units The following illustration shows an existing unit which is outlined in green Use the procedure that follows to add additional waveforms to the unit To Add Waveforms to a Unit 1 Select the unit by clicking its template in the Units view 2 Click Add Wfs above the Waveforms view or Select the Units Add Waveforms to the Selected Unit menu command 3 Move the mouse pointer to the Clusters view The pointer changes to a drawing hand i 150 Offline Sorter Offline Spike Sorting Software Spike Sorting 4 Click the left mouse button and drag a line around the waveforms to add The circled waveforms are added to selected unit Removing Waveforms from Selected Units In the Clusters Views Wave
175. e continuous data If this item is not selected the raw unprocessed continuous data is stored in the NEX file 234 Offline Sorter Offline Spike Sorting Software File Menu e Export Only the Selected Continuous Sources If checked then only the continuous Sources that are checked in the table are exported to the output NeuroExplorer file e Export Unit Templates as Single Waveform Variables Creates a new NeuroExplorer waveform variable for each unit Each variable contains a single waveform at timestamp 0 which is the unit template e Export Time Segments as Internal Variables If checked creates a NeuroExplorer Interval variable for each time segment Each Interval variable will contain a single time range 3 Select a time range of the timestamps to save to the file or accept the default setting where all timestamps are saved to the file A Time Segment can also be used to define the Time Range The Time Range Limit Also Affects checkboxes control whether the Continuous data and Events will also be limited to the specified Time Range If unchecked the default all Continuous data and Events will be exported even if a Time Range is specified You can optionally export a NeuroExplorer waveform variable for each unit s template The template waveforms have timestamp 0 Also you can optionally write out the timestamps for the unsorted waveforms to the NEX file as if it were a sorted unit The NeuroExplore
176. e dan ena nae A E A A 262 5 8 1 Tools Recalculate the Principal Component cccesseececeeeeeeeeeeeeeeeeeaaeeeees 262 Soa VOLS LAI Py AVION a 263 ce Tol Adut GaN Eaa N ae 263 5 84 Tools ihvert WavelonmS ci cessoatesssccuiectasessouerestiouee ta sthiudepnGouleiassauouetaladensdine 263 5 8 5 Tools Re Threshold WavelorME sinc scsvaicacehunsiddetesunaicatetsnseieattedstanctettadvadackseatilascns 263 FB6 Toole intera Seleti iaria oi AEA Ear 264 5 8 7 Tools Remove Short ISI Waveforms simiimsnriisiissii nai 267 20S Tool Romove AMETE orainion a UA KETENE 267 5 8 9 Tools Assign Unsorted to Closest Unit sccisccisssscetasscscsscoseivscctnaesecosseranevagacnsaetenaaies 267 5 8 10 Tools Invalidate Cross Channel Artifacts ccccccccsceeeseeeeeeeeeeeeeeeeseeeeeeaeeeees 267 5 8 11 Tools Invalidate High Amplitude Artifacts 20 0 eccceeeeeeeeneeeeeeeenaeeeeeeenaeeeeeeeeaas 267 So ine ets MAINS Levo osnan 268 te baw MNS AO AGG caria aeai Eara R R OES 269 Bee WE TOS TNS es aga teen ptaha tsa sadaniedl A a benisaaeantaeatd Seeaseeas yacenees 270 BO eS DAE norn CHONG saisan aaka aA Aaaa a 271 Do VUNG MGI cccccrivcnssancepteadesietcuadsinitycncdesiey i EAA ES 272 SAO PUn E O is sae a fincstatied pasa ad scare pate mea paar pa mein p aes eel gee Dane eee tetae et 272 Pe UU ERD KE rea a taste a curd scans aut ta ca acuta seats Lucu 273 TLEAN WOON naa aaa uanactaneaccorpemutenetanacs 273 gee QUC Key Felre nE acc icccinserastascadare
177. e mode Feature vs Feature View Left Click Select nearest point Alt Right Click Displays context menu Right Click Drag Zoom the image both axes together Ctrl Right Click Drag Zoom the image each axis independently Shift Left Click Drag Pan the image SEES Ine PETEERE PI eter ROO Teh Mead MIRC CNSR DRTS capOa nr cad Meee ay fern ee en eT EN Scroll down to find the key combinations that apply to the view currently being used To print the complete three page reference click Print Click OK to close the window and return to Offline Sorter Software Version 4 0 273 5 Menu Options 5 11 3 Quick Batch Reference Click Quick Batch Reference to show the following window eS Sa gt Quick Batch Reference T es ae gt E File Dir ForEachFile Save SaveToNex or ExportToNex SaveAsPlx or ExportToPlx SaveAsP1x0 for ExportToP1x0 SortUsingTPL Invalidate rtifacts Invalidate rtifactsa fter Export WavefornInfo ExportChannelInfo ExportUnitInfo ForEachChannel UpSanple Filter Detect Align Sort ValleySeek2d for ValleySeek ValleySeek3d wh 244 T PEE eee EA This window lists all Offline Sorter batch commands with their arguments See Section 6 9 Batch File Processing on page 313 5 11 4 About Offline Sorter Click About Offline Sorter to view information about the version of Offline Sorter Software that you are currently running 274 Offlin
178. e n features ScanType sets the type of scan to perform for the ForEachChannel Scan command The argument values are ParzenMult DOFMult KMEans StdEM OutlierThresh WfLimit PCAMax KMeansSeed StdEMSeed StdEMBeta TDistSeed ScanStart ScanEnd ScanStep sets the scan range for the ForEachChannel Scan command and the amount to increment the scan parameter for each step ScanNormalize set to 1 to use normalize the sort quality metrics calculated during the scan and used to determine the best sorting ScanStat the sort quality metric to use for Scan Sorting from among p J3 PsF DB Dn Lr ID UseRangeStart UseRangeEnd only consider the portion of the file with timestamps between the Start and End values with the times specified in seconds This range is used for sorting operations and for exporting to PLX PL2 and NEX files Set either the start or the end value to a negative number to cancel a sort range and use the entire file SaveCont set to 1 to have ExportToPLX save the continuous data set to 0 to save only the spike data SaveProcessedCont set to 1 to have ExportToPLX save the upsampled and filtered version of the continuous data set to 0 to save the raw continuous data Savelnvalidated SaveUnsorted set to 1 to have ExportToPLX save invalidated or unsorted waveforms set to 0 to ignore them SaveNexCont set to 1 to have ExportToNEX save the continuous data set to 0 to ignore c
179. e per view toolbar for the Clusters views Selecting a sort method in this menu is equivalent to selecting the sort method using the Sort Method droplist in the Control Grid 5 6 2 Sort Perform Automatic Sorting To quickly and easily explore using different sorting algorithms and settings to sort spikes select the Sort Perform Automatic Sorting menu item which shows this dialog Version 4 0 251 5 Menu Options r Perform Automatic Sorting Which Channels Sort In Current Channel only 2D feature space All Channels in Spike Source SPK Chs 1 16 3D feature space Automatic Sorting Method and Parameters Valley Seeking Parzen Multiplier 1 D O F Multiplier 10 K Means Scan Range of Units for K Means and Std E M 2 to Standard E M Scan Beta 1 D Yalley Seeking Scan Range of Parzen Mult for Valley Seeking 0 5 to 1 5 stepby 0 2 T Dist E M Scan Range of D O F Mult for T Dist E M 10 to 30 stepby 5 Perform Continue Sorting Use existing clusters as the starting point Current Channel Undo Previous Sort Cancel The purpose of this dialog is to bring together capabilities that exist elsewhere in Offline Sorter Software into a convenient location From this dialog the user can easily select which automatic sorting algorithm to use with the radio buttons same as selecting the Sort Method from the Control Grid or using the Sort Change Sort Method menu ite
180. e time range covered by the currently selected time segment is displayed in the Range From and Range To parameters that follow By default the program displays all the waveforms from a selected channel Use this option to display only a subset of waveforms based on a time segment It is also possible to set up how the data file is divided into time segments using the Waveforms Create Time Segments menu command Draw Grid draws a grid in the Waveforms view Draw Thr Draw Threshold draws a horizontal dashed red line in the Waveforms view to show the position of the threshold for the channel Refractory Interspike Interval is the minimum valid interspike interval If there are intervals smaller than the refractory interval Offline Sorter Software displays a red bar to the left of the template display in the Units view that corresponds to the proportion of spikes with ISIs less than the refractory interval See Units View on page 42 Lines between spikes with ISIs less than the refractory interval are also drawn in the Clusters views when that option is enabled The ISI is specified in microseconds For more information see Section 3 7 2D and 3D Clusters Views on page 52 Version 4 0 41 3 Views and Displays ISI Hist Max Interspike Interval ISI Histogram Maximum is the maximum value of the X axis in the Interspike Interval Histogram in the Units View in milliseconds Invalid w f Invalid Waveforms
181. e unique within each Source e g PL2 or within channels of the same Type e g PLX e File Shows Y if the Source was present in the original data file or N if the Source was created within Offline Sorter Software Some operations will create new Sources within Offline Sorter Software such as extracting spikes from continuous data e AssocID Sources may have a temporary association between them for example if spikes are extracted from data in a continuous Source then the spike and continuous Sources are associated Offline Sorter Software may show the data from channels in associated Sources together in the same Offline Sorter channel For example after spike extraction Offline Sorter channel 33 may show both the spikes from the Source with Id 5 and the continuous data from the Source with Id 1 See Section 6 1 Details of Channels and Sources on page 277 e Bits The number of bits in each sample for spike and continuous Sources e MaxvV The voltage in mV at the input to the analog to digital converters that will result in a maximum sample value which depends on the number of bits in each sample Only relevant for spike and continuous Sources e NPW Number of Points in each Waveform For spike Sources this is the number of samples captured in each spike waveform segment e NPre For spike Sources the index of the sample within the spike waveform segment where the threshold crossing occurred e Sort
182. ePartner CinePlex CineTracker CineTyper DigiAmp MiniDigi Offline Sorter OmniPlex PL2 PlexBright PlexDrive PlexSort PlexStim Plextrode Radiant RapidGrid TrackSort and the Plexon logo are trademarks of Plexon Inc Dallas Texas USA Other product and company names mentioned are trademarks of their respective owners Offline Sorter Offline Spike Sorting Software Plexon Inc Publication History April 2015 Version 4 0 Version 4 0 of the Offline Sorter User Guide issued The changes from Version 3 0 include the following Throughout the document additional support is added for managing PL2 files Throughout the document updated screen graphics were incorporated Installation and licensing details have been updated See Chapter 1 Getting Started The terms channel and source are described more fully See Section 2 5 Terminology on page 16 Threshold Scan Graph and Overlaps Analysis views are new in the View menu See Section 3 1 Overview on page 21 Additional information was added for the Sources view in the Info Grid Views dropdown menu See Section 3 2 Info Grid Views on page 25 In the Channel Parameters view several options were added SNR Signal to Noise Ratio LC Filter low cut filter Poles and Cutoff See Section 3 2 3 Channel Parameters View on page 30 In the Time Segments view time segments can be name
183. ear Scan Sorting Range of Units for K Means and Std E M 2 to 7 Range of Parzen Mult for Valley Seeking 0 5 to 1 5 stepby 0 2 Range of D O F Mult for T Distribution E M 10 to 30 stepby 5 And in the Tools Options Stats tab the sort quality statistic that is used to determine the best sorting within the scan can be set Sorting Quality Statistic to use for Scan Sorting Methods Pseudo F Me Normalized There are four different scanning sorting methods that can be selected in the Sort Method drop list in the Control Grid or by selecting the corresponding entry under the Sort Change Sort Method menu item K Means Scan Scans over a range of the number of units as specified in the Tools Options Sort tab Since the K Means method is a semi automatic sorting method Offline Sorter Software chooses the initial cluster centers using one of the two different methods For either method Offline Sorter Software calculates the bounding box of all the waveforms in either 2D or 3D feature space That is it finds the smallest axis aligned box that contains all of the waveform points Then depending on the Seed cluster pattern option in the Tools Options Sort tab it determines the initial cluster centers as follows e Circular the N 1 cluster centers are evenly spaced around the perimeter of a circle that fits into the bounding box The final cluster center is placed at the exact center of the circle e Linear
184. ears on the status bar when a converted channel is opened 3 The waveforms for the four time coincident spikes are concatenated together to form the expanded waveform for the single spike on tetrode channel If each single channel contains 800 microsecond long waveforms the four spikes on the four single channels become a single spike on the tetrode chan nel with a length of 4x800 3200 microseconds Any dropped waveforms are represented as zeros in the corresponding position in the expanded waveform 6 2 3 Loading a Channel Map from a File Prior to Offline Sorter V4 the Channel Map view would permit in place editing of the channel map This turned out to be problematic With Offline Sorter V4 the only way to remap channels is to load a channel map from a file that contains the remapping definition By convention the suggested file extension for channel mapping definition is ofm If an ofm file that contains the channel mapping already exists on disk then to apply that channel map in Offline Sorter Software from the in the Info Channel Map tab right click shortcut menu then select Load Channel Map from a File You can also apply the channel mapping file in a batch command file using the Set ChanMap filename command see Section 6 9 Batch File Processing on page 313 The format of the channel mapping file is as follows e The first line contains the number of Sources that are represented in the file The lines followi
185. echnical Support c ceeeceeeteeeeeeees 15 2 Offline Sorter Updates ensarriar Version 4 0 1 Getting Started 1 1 Installation This section explains how to install the Offline Sorter program files and enable the Offline Sorter licensing function You can obtain the Offline Sorter Software by downloading it from the Plexon website http www plexon com or from the USB flash drive provided by Plexon when you purchased the software Note Version 4 0 of Offline Sorter Software installs on Windows 7 only The installation procedure varies based on the status of your computer If you ordered Offline Sorter Software as part of a complete system including a computer and software supplied by Plexon the Offline Sorter Software is already installed on the computer supplied with your order However we recommend that you check the Plexon website to see if an updated version of the software has been created particularly if a significant amount of time has elapsed since you received your system If you ordered an upgrade to your existing Offline Sorter Software the upgrade can be downloaded from the Plexon website and installed with the upgrade code and upgrade procedure provided by Plexon If you are performing a fresh installation of Offline Sorter Software on a computer that does not currently have this software follow the procedure provided below Note The licensing function is managed by means of Sentin
186. ect to apply the final Template Sorting either to the current channel only or to all channels in a Source after the OK button is pressed The Apply and Undo Previous Sort buttons are useful to explore different parameter values 5 6 4 Sort Apply Template Sorting with Current Templates Selecting this menu item will perform Template Sorting using the current unit templates and tolerances It is equivalent to pressing the i main toolbar button l 5 6 5 Sort Apply Template Sorting with Adaptive Templates Selecting this menu item will perform Adaptive Template Sorting using the current unit templates and the adaptive templates settings see next section It is equivalent to pressing the 3 main toolbar button 5 6 6 Sort Adaptive Templates Settings This menu item brings up the Adaptive Template Settings dialog See Section 4 5 4 3 Applying Adaptive Template Sorting on page 160 5 6 7 Sort Save Sorting to SORT File Restore Sorting from SORT File When examining the sorting on a channel it sometimes useful to play what if games to experiment with sorting the channel using different sorting parameters or sorting methods To facilitate this Offline Sorter Software has features that can save the current sorting i e the current unit assignment of every waveform of a channel to a file that can be later recalled By default these files have a SORT file extension This allows a bookmark to be set on what m
187. ed help files in the directory C Program Files Plexon Inc OfflineSorter V4 Ona 64 bit machine whether you have installed the 64 bit version or the 32 bit version of Offline Sorter Software by default the system places the Offline Sorter program and associated help files in the directory C Program Files x86 Plexon Inc OfflineSorter V4 Note The installation process automatically installs the Sentinel Drivers Sentinel Drivers Version 7 5 8 for Offline Sorter Software Version 4 0 if they are not already installed on your computer Delete the Offline Sorter Software setup file from your desktop 8 If you installed the software from the Plexon USB flash drive Select the Windows Safely Remove Hardware and Eject Media toolbar icon Then remove the Plexon USB flash drive from the USB port and store it in the original box 1 2 Using the Sentinel USB Licensing Key Plexon provides at least one USB licensing key with each Offline Sorter Software purchase Additional keys can be ordered from Plexon For Offline Sorter Software to run with full functionality on a particular computer a licensing key must be plugged into a USB port on the computer If the system does not detect a valid license Offline Sorter Software opens in Demo mode as described in Section 1 4 Demo Mode on page 5 1 3 Licensing Functions Offline Sorter Software contains facilities for examining the licensing status of Offline Sorter and other Plexon products an
188. ee Manual Short ISI Spike Removal below 4 6 4 2 Manual Short ISI Spike Removal If a relatively small percentage of spikes have ISIs less than the refractory period you can manually remove the spike in each pair which is further away from the cluster center First follow the procedure outlined in Section 4 6 4 Removing Spikes with Short Inter spike Intervals on page 190 to get the lines drawn to connect pairs of spikes with short ISIs To remove a waveform from a selected unit 1 Zoom in to the waveform s dot circled below in the Clusters view using the toolbar control buttons 192 Offline Sorter Offline Spike Sorting Software Unit Cleaning 2 Select the unit click at the unit s template display 3 Select Units Remove Waveforms from the Selected Unit menu command 4 Move the mouse pointer to the Clusters view note that the pointer changes pee to a drawing hand gt 5 Press the left mouse button and draw a line around the waveform s dot in feature space while pressing the left button 6 Release the mouse button Version 4 0 193 4 Spike Sorting Step by Step You can repeat this procedure until all the waveforms furthest away from the cluster center with ISIs less than the refractory ISI have been removed ChannelO1a J Info Sal Cluster Display If a waveform is removed by accident you can add it to the selected cluster again within the Clusters view To ad
189. eeeeeeeseneeeeeeneneees 246 5 5 10 Waveforms Invalidate All Waveforms in Selected Unit cccecccesseeeeeeeeneees 246 5 5 11 Waveforms Perform Digital Referencing on Continuous Data ceeee 246 5 5 12 Waveforms Up sample Continuous Datta ccccccceeesceeeeeeeeeseeeeceseeeeeeeeeenaees 248 50 13 Wavefotms Filter Continuous Dalasi 248 Tna e a ISG E I A A E E A A A A 249 ae PU o e E A T E E N A A E A E AE 250 5 5 16 Wavelorms Re Threshold encrena 250 5 5 17 Waveforms Remove Short ISI WaveformS ssseessesesseesseeesssessreesssessreessrnssns 250 5 5 18 Waveforms Auto Zoom Waveform Displays ssssseseessesssissssssrrrssrsrrnsssrsrnsnsreens 250 SES M nde ce sre a a tamu anaatotaeti anemia eantaes 251 get Son Change SOUT MCU WO ssncasaisscssecedcanctensdcccsanumieacuedannpedicussmedesccestateddbuqeneeiescieante 251 562 Sort Panom Automate Soran cs 5 cc sccczsss esocsdeeerd aa a Aa 251 Fics Sa Perform Template SoMa SIE akaa 253 5 6 4 Sort Apply Template Sorting with Current Templates 0 cccsseccsssseeeeesenees 254 5 6 5 Sort Apply Template Sorting with Adaptive Templates ccccsccccsssseeeesenees 254 5 6 6 Sort Adaptive Templates Settings cccccccceceeeeceececeeceeeeeeeeeaeeseeaaeeseeeeseaeeeee 254 5 6 7 Sort Save Sorting to SORT File Restore Sorting from SORT File 00 254 5 6 8 Sort Save Sort Info to TPL File and Sort Sort Using TPL Fle
190. eferencing technique and reference channels Version 4 0 247 5 Menu Options The bottom portion of the display selects which channel is used as the reference channel that will be subtracted from all channels in the target continuous Source Three different methods of specifying the reference channel are supported An existing channel Some recording techniques acquire a channel for the sole purpose of using it as a digital reference channel This option allows specifying both the continuous Source and the Channel to be used as the digital reference The reference channel can be in any continuous Source that has the same digitization frequency as the target Source CAR Common Average Referencing Allows specifying a number of channels from any continuous Source that has the same digitization frequency as the target Source whose signals will be averaged together to form the reference channel The average is taken sample by sample e g if 4 channels are specified then the i th sample in the reference channel signal is just the arithmetic average of the i th sample from each of the 4 channels When this option is specified Offline Sorter Software will create a new continuous Source called CARMon that contains a single channel named CARMon to contain the computed reference signal This CARMon Source Channel is treated mostly as a normal continuous channel in Offline Sorter Software it can be selected and inspected in
191. either single electrode stereotrode or tetrode and so Offline Sorter Channels displaying data from a Source may be considered to have the same Data Type as the Source The format of the Channel Map tab has one line per Offline Sorter Channel The Raw channel number s that are associated with the Offline Sorter Channels appear on the line as shown in the following illustration ee _ For Source Eae Chs 1 ai oo 6 7 8 5 h i aio Tabl on i aki ah iat Mini m wih 286 Offline Sorter Offline Spike Sorting Software Channel Mapping The columns in the Channel Map tab are e Left Margin Number The Offline Sorter Channel number e Name The name of the Offline Sorter Channel e Raw Ch s These columns represent the Raw spike continuous channel numbers from the neural data file that are associated with this Offline Sorter Channel If the Data Type is Stereotrode or Tetrode these columns display two or four Raw spike continuous channel numbers If the loaded file type supports channel re mapping these entries are droplists that allow selecting a different Raw channel number Note Offline Sorter Channel numbers always start numbering at 1 Usually Raw channel numbers also start numbering at 1 but occasionally some file formats start numbering Raw channels at 0 In particular the continuous channels from PLX and PL2 files start numbering at 0 There is always a current Channel Map in effect If no chan
192. el system drivers and a Sentinel licensing key The licensing key is not required for software installation but a valid Sentinel licensing key must be plugged into a USB port of a computer in order to run all functionality of the software There are two versions of Offline Sorter Software The OfflineSorterv4w7 folder is for 32 bit machines and the OfflineSorterx64v4w7 folder is for 64 bit machines Follow this procedure to perform a fresh installation of the software 1 Exit all currently running applications 2 Insert the Plexon USB flash drive containing the Offline Sorter setup application into an open USB port on your computer Then go to Step 3 or Download the Offline Sorter Software either the 32 bit or 64 bit version from the Plexon website http www plexon com Then go to Step 4 3 Open the applicable directory either OfflineSorterv4w7 or OfflineSorterx64v4w7 and navigate to the applicable setup file either OFSv4w7Setup exe for 32 bit machines or OFSx64v4w7Setup exe for 64 bit machines 4 Copy the setup file to your desktop Double click the setup file on the desktop The Offline Sorter Setup screen will appear Offline Sorter Offline Spike Sorting Software Using the Sentinel USB Licensing Key 6 Follow the prompts on the setup dialogs to complete the installation The installation proceeds as follows e Ona 32 bit machine by default the system places the 32 bit Offline Sorter program and associat
193. emplate Sorting proceeds through the file With the right choice of the Adaptive Template Weight and Threshold parameters see Section 6 5 4 Adaptive Templates Algorithm on page 302 the templates can continually adjust to slow shifts in the unit waveforms perhaps caused by a settling electrode over the course of the experiment thus tracking the units as they drift 160 Offline Sorter Offline Spike Sorting Software Spike Sorting To initiate Adaptive Template Sorting select Sort Adaptive Templates Settings or click the ae button on the toolbar The Adaptive Templates Settings window appears Adaptive Templates Settings ial Adaptive Template Parameters V Apply Changes Immediately Weight p o la Update Threshold 20 Undo Previous Sort Sort with Adaptive Templates Done By default whenever the Weight or Update Threshold value is changed using this window the Adaptive Template Sorting is applied to the clusters in the current channel If the results are not acceptable the Undo Previous Sort button will revert the sorting same as pressing the Undo button in the toolbar Unchecking the Apply Changes Immediately checkbox will cause the Adaptive Template Sorting will to be applied only when the Sort with Adaptive Templates button is pressed If the Adaptive Template Sorting parameters have already been set they can be quickly applied to the current channel by selecting Sor
194. en performed by Offline Sorter Software For more information see Section 4 8 1 Saving Timestamps and Waveforms on page 205 2 4 2 1 Exporting to New PLX or PL2 For any of the data formats that Offline Sorter Software can read it can write the spike data out as a PLX file With this feature Offline Sorter SoftwareSorter Software can effectively translate any format data file into a Plexon PLX file The Export to PL2 capability is only available when a PL2 file has been loaded The system writes a new PL2 file that may include new Sources that were created within the Offline Sorter Software For more information see Section 4 8 1 Saving Timestamps and Waveforms on page 205 2 4 2 2 Exporting to NeuroExplorer Offline Sorter Software can export the timestamps of sorted waveforms into a NEX file for use with NeuroExplorer Offline Sorter Software can also export waveforms and continuous data For more information see Section 4 8 2 Exporting to a NEX File on page 206 14 Offline Sorter Offline Spike Sorting Software Offline Sorter Outputs 2 4 2 3 Exporting Per Waveform Data To accommodate later analyses of the data Offline Sorter Software can write out selected information about each waveform into either a text or a MATLAB file or into an Excel spreadsheet For a given waveform any of the calculated feature values can be exported along with the unit assignment timestamp raw waveform voltage values
195. ency Hz shows the frequency in Hz of the timestamp clock Waveforms Channels shows the total number of spike channels in the file Data Type indicates whether the data type is Single Stereotrode or Tetrode A D Frequency Hz shows the frequency in Hz of the analog to digital A D conversion while the data was collected Waveform Length usec indicates the number of clock ticks at the analog to digital frequency of data for each spike waveform Prethreshold usec indicates the number of clock ticks collected prior to the triggering event threshold crossing Number of Points per Waveform indicates the number of samples taken per spike waveform waveform length A D frequency Total Number of Waveforms indicates the total spikes waveforms in the file Peak Voltage at ADC input mV shows the voltage at the ADC input that will produce the maximum analog to digital converter count for spike channels A D Resolution bits is the resolution of the analog to digital A D converter for the spike channels Version 4 0 33 3 Views and Displays Units Total Number of Units the total number of units on all channels for the current file Number of Channels with Units indicates the number of channels that have gt 0 units e Avg Units per Active Chan is the value in Total Number of Units divided by the value in Number of Channels with Units Cont
196. ent for each waveform Semi Automatic Clustering Methods With these methods the user specifies a number of units and starting cluster centers or template waveforms and the algorithms then assign waveforms to the units Automatic Clustering Methods With these methods the number of clusters is decided algorithmically and the waveforms are assigned to the clusters automatically On a given channel any or all of these methods can be used e Section 4 5 2 Manual Clustering using the Contours Method on page 146 e Waveforms are manually selected in feature space in the Clusters views by drawing an arbitrary shape around a visible cluster e Section 4 5 3 Manual Clustering using the Waveform Crossing Method on page 153 e Waveforms are manually selected as belonging to a unit or cluster by crossing a bundle of waveforms in the Waveforms view with the mouse e Section 4 5 4 Semi Automatic Clustering using Templates on page 157 e The user selects an individual waveform to serve as the template for the unit waveforms are then assigned to clusters using a template matching algorithm e Section 4 5 5 Manual Clustering using the Boxes Method on page 163 e The user places a pair of boxes for each unit on the Waveforms view and any waveforms that intersect both boxes are sorted into that unit e Section 4 5 6 Manual Clustering using the Lines Method on page 165 The user draws one or more line segments
197. enu item is not available unless the current channel has continuous data 248 Offline Sorter Offline Spike Sorting Software Waveforms Menu 5 5 14 Waveforms Detect Offline Sorter Software can also perform waveform detection on all the spike continuous channels in a file Use the following procedure to set up waveform detection To initiate waveform detection 1 Click Waveforms Detect The Waveform Detection window appears r Maemo D All Channels in Continuous Source Set Waveform Detection Options SPKC Chs 129 192 Thresholds On Raw Signal Amplitude Only Use Current Thresholds Use a Constant Threshold ofADCRange microVolts ADC Counts 5 00 83 31 1638 D Use Standard Deviations from Mean of Peak Heights Histogram Use MAD 2 70 sigmas enter negative for a negative threshold 2 In the Which Channels area click a channel selection To perform waveform detection on the current channel click Current Channel only To perform waveform detection on all channels in a continuous Source click All Channels in Continuous Source and select the desired Source from the droplist 3 To open the Waveform Detection Options window click Set Waveform Detection Options For more information on detector and waveform options see Section 4 2 6 Specifying the Waveform Detection Options on page 120 4 In the Thresholds area click one of the following detection thresholds e Use Cu
198. eplexQuickSta 09 54 18 24 Reading neural data file C Program Files Plexon Inc CinePlex Temp CineplexQuickSt 09 54 18 29 Reading video data file C Program Files Plexon Inc CinePlex Temp CineplexQuickSta 09 54 18 43 Creating Frame Markers from timestamps in avi File 09 54 18 43 Creating coordinate markers from strobed events Right clicking the mouse in the Messages window will display a right click menu with the following items A 16 Offline Sorter Offline Spike Sorting Software Erase This item clears the window of all messages e Pause This item stops the logging of messages e Show Debug Messages This item is a toggle to show or hide debug messages e Select and Copy All This item allows you to copy all of the messages to another application such as a word processor e Save Log to File This item allows you to save the messages to a log file e Mail Log to Plexon This item allows you to send the message log to Plexon for troubleshooting purposes A 3 5 Right click Menus Most windows have right click menus that control their behavior and options To open a right click menu place the cursor inside a window and click and release the right mouse button The right click menu appears where you click To select a menu item move the cursor over it and click the left mouse button A 3 6 Current Selections In grid based windows the currently selected item always appears with a gt gt or gt in the left col
199. epresentable voltage for spikes and thus be cut off or clipped This again results in information loss 116 Offline Sorter Offline Spike Sorting Software Waveform Acquisition From Continuously Recorded Data F Adjust Gains Channel 28 FP12 0 Waveforms 8575 Continuous Samples Spikes Full Scale Voltage at Probe mV Peak Voltage at ADC Input mv 2500 000 A D Resolution bits 16 Preamp Gain 1 Voltage Resolution at Probe mV bit 0 000076 Total Gain 1000 Optimize Continuous Full Scale Voltage at Probe D Peak Voltage at ADC Input mV 2500 000 A D Resolution bits 16 PreampGain 1 i TotalGain 2000 Set Gains on a Range of Channels Lx Cancel Voltage Resolution at Probe mV bit 0 000038 For example in the above screenshot the largest voltage value in any waveform that is present in the continuous data signal is 1 250 mV But this voltage value represents only half of the full scale voltage range available for extracted spikes 2 5 mV Thus all extracted spikes will be small and some of the dynamic range for spikes will be wasted This situation can be changed by altering the gains until the two Full Scale Voltage at Probe values in this dialog box are more closely matched but such that no clipping of the signal occurs If you press the Optimize button the system will attempt to adjust the gains automatically so as to achi
200. ered for executable files e Arguments This is a list of all the arguments to be passed to the file in the Command area if any The browse button opens the standard Open dialog box filtered for all files When applicable the symbol 1 can be used to denote the current document e Initial Directory This contains the path of the file in the Command area The path allows the system to find the file and execute it The Browse button opens the standard Browse For Folder dialog box Procedure for Customizing the Run Menu 1 From the Window menu select Customize and click the Run Menu tab 2 To add a menu item click the New icon enter the command in the Command text box enter any arguments in the Arguments text box and set the applications directory in the Initial Directory text box In each case you may use the appropriate Browse button at the right of the text box The Browse button for the command opens the standard File Open dialog box filtered for executable files The Browse button for the arguments opens the standard File Open dialog box filtered for all files Then Browse button for the initial directory opens the Browse for Folder dialog box To delete a menu item select it and click the Delete button 4 To reorder the menu items select a menu item and use the Up and or Down arrow buttons to reorder A 4 5 Options Customization An illustration of the Options tab follows r Customize Toolbars Commands Keyboa
201. erforming waveform detection on a continuous Offline Sorter Channel will actually result in a new spike Source being created within Offline Sorter Software to contain the resulting spikes The new spike Source is associated with the continuous Source that provided the continuous data for waveform detection as can be seen by the values shown in the AssocID column in the Sources view After waveform detection the continuous Source is still marked with a C gt and the new spike Source SPK_SPKC in the screenshot below is marked with an S gt in the Sources view Version 4 0 281 6 Reference Topics SR ae eee 1 SPK Spike 164 i 40000 7 Cont 65 1281 40000 4 Cont 129 1921 40000 5 4 FP Cont 193 256 1 10008 Spike 129 192 1 40000 1 2500 0000 a i a y F 2500 0000 65 128 Y 1 16 2500 0000 Yy N 8 T 129 192 16 2500 0000 2500 000032 8 1 32 Similarly performing low cut filtering on continuous data in a channel will result in a new continuous Source being created to hold the filtered version of the continuous data Since low cut filtering is applied on a channel by channel basis each Offline Sorter Channel remembers whether it should show the non filtered data from the original Source or the filtered data in the new Source when that channel is selected When a Offline Sorter Channel is selected the Timeline view will be configured to show the correct data from either the spi
202. es However if this value is too large it can slow performance Buffer Size for Continuous Data specifies the size of the memory buffer that Offline Sorter Software uses for holding the data from continuous data channels If it is not desired to work with continuous data channels or if it is acceptable to wait for continuous data to be dynamically read in after a channel is initially loaded set this to a small value 6 11 4 Ignoring Continuous Data Channels Plexon PLX or PL2 files often contain a large amount of continuous slow channel data that can be a significant fraction of the total file size By default Offline Sorter Software reads in this continuous data when the file is opened If continuous channel data is not desired instruct Offline Sorter Software to bypass reading the continuous data altogether by checking the Disable loading continuous data channels checkbox under the Continuous Data section on the General tab of the Tools Data Import Options dialog box CAUTION Continuous Data Channels Quietly Ignored When Disable loading continuous data channels setting is checked continuous data channels are quietly ignored Offline Sorter Software gives no indication that these continuous channels even exist in the data file and they are not saved during a Export to New PLX operation Moreover Offline Sorter Software retains this setting between startups If this setting is checked Offline Sorter Software ignores continuous
203. es this threshold either in the current channel or in all channels within a specified spike Source The artifact threshold can be specified as either a percentage of the ADC range in microvolts or in ADC Counts Additionally the tool is also able to invalidate other spikes within a time window following an artifact detection Any spikes occurring within the specified number of milliseconds following an artifact threshold crossing will also be invalidated whether these subsequent spikes exceed the artifact threshold or not 5 8 12 Tools Density Leveling This tool is somewhat experimental and should be used carefully The goal of the tool is to emphasize the units that fire rarely and so have few waveforms in the unit by de emphasizing units that have high firing rates and so have large numbers of waveforms in the unit Its operation is best visualized by picturing the Surface view see Section 3 8 Surface View on page 69 Each column in the Surface view has a height determined by how many waveforms are in that column s bin This tool will seek to reduce the height of the tallest columns by randomly invalidating spikes that fall into those columns bins The probability of randomly invalidating a spike in a bin is proportional to the number of spikes that fell into that bin the height of the column Thus tall columns are reduced much more than short columns This produces a more level picture of the waveforms in th
204. eshold a SNR calculation is done following the extraction and the SNR value along with the number of spikes extracted is plotted on the graph Thus the graph can be built up by changing the threshold and pressing the Detect button to extract spikes Like all the various Graph views in Offline Sorter Software hovering the mouse over a point will show the numerical values associated with that point as shown in the screenshot The SNR is computed as the ratio of sigma squared within signal sigma squared within noise where the signal is taken to be all samples within extracted spikes and the noise is all samples outside of extracted spikes Scanning Over a Range of Thresholds Selecting Scan Thresholds from the right click menu will show the Threshold Scan dialog 98 Offline Sorter Offline Spike Sorting Software Overlaps Analysis View Step the threshold value over a range performing a spike extraction at each value Specify the threshold value as Using this dialog you can systematically step the threshold from a starting value to an ending value and perform a waveform extraction at each threshold value resulting in a point in the Threshold Scan Graph at each threshold This produces a graph as shown in the screenshot in the previous section Note that for large thresholds far away from zero either positive or negative the extraction produces few spikes but the SNR for those few spikes is very
205. et the number of cluster centroids used in this initial sort by using the Tools Options Sort dialog in the Number of seed clusters for T Distribution algorithm edit box The initial cluster centers given to the K Means algorithm are evenly spaced along a diagonal line through the bounding box in feature space that contains all of the points to be sorted References Shoham S Fellows M and Normann R Robust automatic spike sorting using mixtures of multivariate t Distributions Journal of Neuroscience Methods 127 2 111 122 2003 Figueiredo Mario and Jain Anil Unsupervised Learning of Finite Mixture Models IEEE Transaction on Pattern Analysis and Machine Intelligence vol 25 pp 1150 1159 Sep 2003 6 5 9 Partial Sorts Normally for the automatic sorting methods initiating a Sort on a channel deletes all existing units and creates new units according to the algorithm However when the sorting algorithms are run when not all the waveforms are visible in the Clusters views because the Use Only Unsorted Waveforms or Use Only Currently Visible Waveforms options are enabled and the waveforms being displayed are being limited by the Waveforms view setting or by the Control Displays checkbox on the Timeline view the behavior of the sorting algorithms changes All existing units are preserved and the sorting algorithm is only allowed to run on the currently visible waveforms This generally results in some number of new units in ad
206. et to 0 For further discussion see Section 6 3 Stereotrode and Tetrode Data on page 291 296 Offline Sorter Offline Spike Sorting Software Features Available for Sorting 6 4 1 Principal Components The projections onto the first eight principal components can be plotted on any axis of the Clusters views By default the principal component vectors are calculated using all the valid waveforms of the current channel and all time points in the waveform between Sort Start and Sort End For more information see Section 4 4 5 Adjusting the Sort Range on page 142 For example waveforms projected onto the first two principal components appear as follows when viewed in the 2D Clusters view y 2D Clusters 3D 3D Clusters Surface Waveform Detection The dashed lines are the X and Y axes representing the zeroes of PC1 and PC2 The points representing the lowest amplitude waveforms have principal component projections near zero i e near the intersection of the two axes You can recalculate the principal components using subsets of waveforms e g only valid waveforms only unsorted waveforms only waveforms within a time range or using only the points specified by the Sort Start and Sort End parameters in the Control Grid The various options appear in the Tools Recalculate the Principal Components menu command which displays the following window Version 4 0 297 6 Reference Topics 7 Recalculat
207. etes You can set the Max Iterations parameter in the E M Sorting section of the Sort page under the Tools Options dialog box There is a parameter in the Control Grid labelled D O F Mult that appears when the T Distribution E M sorting method is selected This parameter controls the convergence properties of the algorithm for details see Section 6 5 8 T Distribution E M Algorithm on page 305 The first step of the T Distribution sorting algorithm is actually to do a K Means sort using a preset number of seed cluster centers The E M algorithm can then combine clusters together as the algorithm progresses to arrive at the optimal number of final clusters You can set the number of seed clusters used initially in the E M Sorting section of the Sort page under the Tools Options dialog box The T Distribution sorting algorithm never produces more clusters than the initial number of seed clusters You can clean up the clustering results after the sorting is completed by adding or removing waveforms from each unit e either in the Waveforms view e See Adding Waveforms to Selected Units in the Waveforms View on page 154 and e Section 4 6 3 Cleaning the Selection on page 187 e or in the Clusters views 178 Offline Sorter Offline Spike Sorting Software Spike Sorting e See Adding Waveforms to Selected Units in the Clusters Views on page 150 and e See Removing Waveforms from Selected Unit
208. eve the best possible results for extracting spikes from continuous data Note For versions of Offline Sorter Software prior to 3 0 waveform extraction was done in a d count space in that the threshold was applied to the raw a d values and the raw continuous a d values became the raw spike a d values verbatim This would generally avoid the clipping and dynamic range under utilization problems described above but could make it nearly impossible to recover actual voltage values in the extracted spike waveforms Starting with Offline Sorter Software 3 0 the waveform extraction is now done in Voltage at the probe space where if a waveform peak was 1 0 mV at the probe in the continuous data the waveform peak will also be 1 0 mV at the probe in the extracted spike If the gain settings need to be changed for many channels pressing the Set Gains on a Range of Channels button will bring up a simple dialog where the range of channel numbers can be specified If the OK button is pressed those channels will have their per channel gain settings set to be identical to the channel being shown in the Adjust Gains dialog Version 4 0 117 4 Spike Sorting Step by Step 4 2 5 Selecting a Digital Filter Low cut or High cut Offline Sorter Software can filter the signal from an active channel by using low cut digital filters The filters available in the program are Butterworth Bessel and Elliptic IIR filters up to 12 order
209. ew Offline Sorter Software can display a graph of selected sort quality metrics as a function of time segments The values plotted are the same as those shown in tabular format in the Section 3 2 5 Time Segments View on page 34 This view can reveal episodes of poor sort quality over certain periods in the file To display the Sort Quality vs Time Segment Graph View Select View Sort Quality vs Time Segment Graph view menu command or press the 4 button on the toolbar The vertical axis has arbitrary scaling each plotted sort quality metric is scaled independently so that the entire range of sort quality metric values fits vertically on the plot The horizontal axis is each Time Segment See Scan Graph View on page 25 for details on how to customize the display by showing a different selection of sort quality metrics or plotting them with different colors or symbols 3 21 Threshold Scan Graph The Threshold Scan Graph view displays a graph of the number of spikes found and the signal to noise ratio SNR as a function of the threshold position An entry is made in the graph each time spikes are extracted on a channel containing continuous data To display the Threshold Scan Graph Select the View Threshold Scan Graph menu command or press the button on the toolbar Version 4 0 97 3 Views and Displays When the Threshold Scan Graph is showing every time a spike extraction is done at a specific thr
210. ew file name for the file You cannot overwrite the original file with this operation Offline Sorter Software supports a flexible means to save timestamps and waveforms into a PLX file No matter how the waveforms are read into Offline Sorter Software from any supported file format or after waveform extraction has been performed the waveforms can be saved into a PLX file Select the File Export to New PLX menu command to bring up the following dialog box 7 Barone POH C PlexonD ata pl2 test files all data 64 channels 01 What to Export Export Invalidated Waveforms V Export Unsorted Waveforms V Export Continuous Data V Export Up Sampled Filtered Continuous Data _ Export Only the Selected Continuous Sources Chs 65 128 129 192 193 256 Time Range to Export Entire File Only Data from within the Time Range From sec 9 To sec O Only Data from within the Time Segment 1 Time Range Limit Also Affects Continuous Range of Samples within Waveforms to Export All Samples Only the Samples From Tick 0 ToTick 32 File Select the name of the output file Click the Browse button to bring up a file browser Export Invalidated Waveforms If this item is selected Offline Sorter Software still writes waveforms that have been invalidated to the output PLX Version 4 0 231 5 Menu Options file If unselected default Offlin
211. f the box circled in previous illustration The Unit to which the waveform is assigned is illustrated by a colored bar circled in previous illustration In the earlier example the selected Waveform 202 on this channel occurred at time t 1 96755 seconds and is assigned to the green unit Unit b Note In most cases for correctly sorted units the unit template average waveform that best matches the waveform minimum sum of squared difference should also be the unit to which the waveform is assigned That is the best match and the colored bar should be in the same template box 4 7 2 Examining Sorting Statistics There are two methods to examine sorting statistics Each one is described in detail in this section 4 7 2 1 Info Grid Views Statistics The Stats view under the Info tab contains more detailed statistical information about the sorting performed on a channel The Sorting Summary View detailed 200 Offline Sorter Offline Spike Sorting Software Examining the Sorting on page 84 provides a convenient place to look to see a more concise summary of the most important sorting quality statistics Sorting Statistics for Channel 148 Features PC 1 PC2 PC3 Multivariate ANOVA Not Including Unsorted Waveforms as a Group _ In 2D Cluster Space H F 8 8888 i 46 6529 i P 3 97725e 074 in 3D Cluster Space F 12 14755 47 0379 p 2 30573e 110 20 3D J3 746 2556 7 24072 Pseudo F 1
212. fline Sorter User Guide issued September 2003 Version 2 4 of the Offline Sorter User Guide issued April 2003 Version 2 3 of the Offline Sorter User Guide issued vii Offline Sorter Offline Spike Sorting Software viii Offline Sorter Offline Spike Sorting Software Plexon Inc Contents Publication History Chapter 1 Getting Started Installation 2 Using the Sentinel USB Licensing Key 3 Licensing Functions 3 Demo Mode 5 Off line Sorter Technical Support and Updates Offline Sorter Technical Support 5 Offline Sorter Updates 5 Chapter 2 System Overview Version 4 0 Offline Sorter Uses 8 Offline Sorter Inputs 9 Sorting Techniques 10 Offline Sorter Outputs 14 Printing Results 14 Saving Results 14 Exporting to New PLX or PL2 14 Exporting to NeuroExplorer 14 Exporting Per Waveform Data 15 Exporting Per Channel Data 15 Exporting Per Unit Data 15 Reproducing Results 15 Export Sort Infoto TPL 15 Sort Using TPL file 15 Sort Using NEX file 15 Execute Batch Command File 16 Terminology 16 Spike Waveform Segments 16 Continuous 16 Digital 16 Fast and Slow 16 Channel 16 Source 16 5 Offline Sorter Screen Layout 17 Toolbars 17 Status Bar 17 Views 18 Chapter 3 Views and Displays 19 Overview 21 Info Grid Views 25 Sources View 27 Channels View 29 Channel Parameters View 30 File Summary View 32 Time Segments View 34 Sort Details View 35 Events View 36 Stats View 36 PCA View 36 Scan View
213. fline Spike Sorting Software 2D and 3D Clusters Views The display of the Look At icon can be turned on and off using the 3D Look At Icon checkbox in the Tools Options Display tab The color of the Look At icon can be changed in the Tools Options Colors tab The look at point can be changed in two ways e Manually positioning e Positioning it to the center of gravity of clusters To manually position the Look At point 1 Hold down the Shift key then click and hold the left mouse button 2 The pointer changes to a hand Mouse movement then translates the eyepoint in screen space That is moving the mouse in the left right direction translates the Look At point in the screen x direction and moving the mouse up down translates it in the screen y direction 3 To move the Look At point nearer or farther from the eye hold down the SHIFT key then click and hold the right mouse button The pointer changes to a hand Moving the mouse up and down translates the Look At point in the screen z direction TIP Moving the Look at Point To get accustomed to manually moving the Look at Point think of the hand grabbing and pulling the enclosing 3D grid box To position the Look At point at cluster centers Press the C or L key to position the Look At point at the center of gravity CG of the unit clusters and make a selection by cycling through each assigned unit cluster with a key press The center of gravity of all points and the ce
214. for removing them To Draw Lines in the Clusters view between spike pairs with interspike intervals less than the Refractory ISI 1 Select Draw lines showing detected small interspike intervals in the Refractory Period tab on the Tools Options dialog box 2 After zooming in to the cluster in question using the zoom aw E A E and move F gt od i control buttons on the toolbar the Clusters view might then look like this 4 6 4 1 Automatic Short ISI Spike Removal Offline Sorter Software provides a tool to automatically remove waveforms with ISIs below the refractory period Selecting Tools Remove Short ISI Waveforms from the main menu removes waveforms from the clusters according to the following 2D algorithm For each unit 1 Calculate the center of gravity of points in that unit This gives a point in 2D feature space that is near the center of the cluster Version 4 0 191 4 Spike Sorting Step by Step e For each pair of waveforms with ISIs below the Refractory Period calculate which member of the pair is furthest from the center of gravity point for the unit 2 Remove that waveform from the unit Note After this tool runs short ISI waveform pairs can still exist if a particular waveform was within the refractory period of more than one other waveform If needed re run the tool to get rid of all short ISI waveforms It is also possible to manually remove spikes with short ISIs from units S
215. for the application Click a toolbar checkbox to have it appear in the main application window e New This button opens the New Toolbar dialog box You can use this feature to create a custom toolbar for commands you frequently use Enter a toolbar name in the Toolbar name box Rename If you have selected a toolbar that you previously defined click Rename to change the name of the toolbar The Rename Toolbar dialog box displays e Delete If you have defined a new toolbar and selected it you can click Delete to remove that toolbar There are no default values for new toolbars A 18 Offline Sorter Offline Spike Sorting Software you define You cannot delete the standard toolbars A confirmation dialog box displays Reset If you have selected a standard toolbar click Reset to restore the toolbar to its default contents If you have dragged new buttons to a toolbar click Reset to restore the default version of the toolbar A confirmation dialog box displays A 4 2 Commands Customization The Commands tab is used to customize which commands are available in toolbars An illustration of the Commands tab follows Customize Toolbars Commands Keyboard Run Menu Options To add a command to a toolbar select a category and drag the command out of this dialog box to a toolbar Categories Commands Edt O New Project ba View gt Open Project Animate Save Project Save Project As
216. forms can also be trimmed away from existing units To Remove Waveforms from a Unit 1 Select the unit by clicking its template in the Units view xX Add Unit Delete Unit Add Wi or select the Units Remove Waveforms from the Selected Unit menu command Version 4 0 151 4 Spike Sorting Step by Step 3 Move the mouse pointer to the Clusters view The pointer changes to a drawing hand f 4 Click the left mouse button and drag a line around the waveforms to remove The circled waveforms are removed from the selected unit Retaining Waveforms In Selected Units In the Clusters Views Offline Sorter Software also includes a reciprocal function that retains the selected waveforms and removes all the unselected waveforms This function is similar to previous removal function except that all selected waveforms are retained and the rest are removed from the cluster To Remove All Unselected Waveforms from a Unit 1 Select the unit by clicking it s template in the Units view 2 Select the Units Remove All Except Selected Waveforms from the Selected Unit menu item or click the corresponding toolbar button 3 Move the mouse pointer to the Clusters view Bi The pointer changes to a drawing hand 4 Click the left mouse button and drag a line around the waveforms to retain The uncircled waveforms are removed from the selected unit Deleting Units Units can be removed entirely To Delete
217. forms that should NOT be removed from the currently selected Unit This is equivalent to pressing the 2 button on the main toolbar 5 7 10 Units Automatically Recalculate Unit Templates after Each Change This is a checkable menu item When checked Offline Sorter Software will recalculate the unit templates after any change that affects which waveforms are in the unit See Section 4 5 4 1 Managing Unit Templates and Fit Tolerances on page 158 for details 5 7 11 Units Calculate Unit Templates from Existing Units When selected this will recalculate the unit templates immediately See Section 4 5 4 1 Managing Unit Templates and Fit Tolerances on page 158 for details 5 7 12 Units Automatically Recalculate Fit Tolerances after Each Change This is a checkable menu item When checked Offline Sorter Software will recalculate the unit Fit Tolerances after any change that affects which waveforms 260 Offline Sorter Offline Spike Sorting Software Units Menu are in the unit See Section 4 5 4 1 Managing Unit Templates and Fit Tolerances on page 158 for details 5 7 13 Units Calculate Fit Tolerances from Existing Units When selected this will recalculate the unit Fit Tolerances immediately See Section 4 5 4 1 Managing Unit Templates and Fit Tolerances on page 158 for details Version 4 0 261 5 Menu Options 5 8 Tools Menu The Tools menu contains entries for performing various operat
218. ge 175 sorting algorithm which scans the number of clusters over the range specified Offline Sorter Software automatically sets the cluster center positions by using the same procedure previously described for the Scan number of clusters for K Means and the same caveats apply Scan Outlier Threshold This scan type runs the Remove Outliers tool See Cleaning the Selection on page 187 scanning the Outlier Threshold over the range specified by the Start End and Step By values At each step the sorting resets to the initial state that is the effects of the Remove Outliers tool are undone after each step However when one clicks Re Sort Using This Value or double clicks on a scan table line remember to undo the action before it is repeated or the Remove Outliers tool is applied to the results of the last operation which is not the same initial condition that was in effect during the initial scan Scan Waveform Limit for Valley Seeking This scan type runs the Automatic Clustering using Valley Seeking Method page 176 sorting algorithm while stepping the waveform limit over the range specified The waveform limit which can be set in the Tools Options Sort dialog box limits the number of points used by the Valley Seeking sorting algorithm A typical scan range for this type of scan might be 1000 to 10000 in steps of 1000 This is useful way to get a feeling as to how the number of points can affect the values of the sort q
219. ght by default Show Tetrode Min Max Electrode Combinations this option is similar to the Show Tetrode Electrode Combinations option in that it shows the combinations of electrodes but this version shows both the Peak Height largest positive voltage and the Valley Height largest negative voltage features simultaneously for a total of 12 plots Common Functionality In general the plots in the Feature vs Feature view behave the same as the 2D Clusters view plot responding to the Add Remove Waveforms Units buttons Version 4 0 83 3 Views and Displays and menu items the Waveforms view selection and the zooming panning toolbar buttons in the same way For example the following mouse functions from the 2D Clusters view are available e Left Click selects the nearest waveform point highlighting that point on all displays and in the Waveforms view e Control Left Click while in Contour sorting mode adds a new unit e Shift Left Click pans the display e Right Click and hold zooms the display in and out e Alt Right Click displays the context menu e Rubber banding with the middle mouse button zooms the display Additionally Double click any sub box to bring up the full size 2D Clusters view with the corresponding features on the X and Y axes 3 14 Sorting Summary View The Sorting Summary view can be used to quickly get an overview of the results of sorting a channel The Sorting Summary view is intended to be
220. ginal batch command file name Offline Sorter Software also has a convenience command File View Log from Last Executed Batch Command File that will quickly show the log file from the last batch execution in the current editor Version 4 0 235 5 Menu Options 5 1 13 File Print Print Preview Print Setup Offline Sorter Software can print the contents of most of its views Select File Print or File Print Preview to print or preview the currently active window If printing for the currently active view is not supported these menu selections will be ignored If the computer has Adobe PDF Distiller installed the File Print All Sort Summaries offers a means to easily get the Sort Summaries for all channels in a file into a single PDF document Simply select Adobe PDF as the printer in the Print dialog and type the PDF file name The colors and the Clusters view point size for printing can be different from the values used for display Select the values to use for printing in the Printing Options tab in the Tools Options dialog box By default the printing colors are the same as the display colors except that the background color is set to white Some of the display colors do not print with high contrast on some printers The 2D Clusters view has an option to dump the display to a Windows metafile which can then be imported into most graphics programs To do so from the right click menu on the 2D Clusters
221. haracteristic can enable E M algorithms based on T Distributions to better handle outliers in the data set 2 The algorithm has built into it a way to automatically decide on the number of clusters This feature is agglomerative in nature as the algorithm starts with a large number of clusters and combines them together as the algorithm proceeds so as to minimize a penalized likelihood function As discussed in the Shoham reference the algorithm does not perform well when using the theoretical value for the number of parameters per component Offline Sorter Software introduces a multiplier on this theoretical degrees of freedom value that can be adjusted Empirically values of around 20 30 produce the most pleasing clusters Smaller values generally lead to too many clusters larger values lead to too few The Scan feature in Offline Sorter Software can perform sweeps over this sorting parameter so its affect on sorting performance can be investigated The algorithm must be started with an existing clustering of the data generally with too many clusters defined as the algorithm can agglomerate clusters together as it proceeds The initial clusters can either be taken from the Version 4 0 305 6 Reference Topics currently defined units See Sort Perform Automatic Sorting on page 251 or they can be generated automatically To automatically generate the starting configuration the K Means algorithm is used You can s
222. he full height lines are the events the shorter lines are spike waveforms ply 65 Zoom Out Zoomin lt You can quickly adjust the settings in the Show and Display Color columns by using Set As Top Row in the right click context menu The Randomize Colors entry in the context menu generates a random color for each different event Several of the data formats other than Plexon PLX and PL2 files have an information type comparable to events e g Datawave Markers Wherever Version 4 0 137 4 Spike Sorting Step by Step possible these information records are translated into events when the data file is loaded Plexon PLX and PL2 files also have the concept of strobed events Strobed events have an event channel number of 257 but have an additional data fields that stores a data word In Offline Sorter Software there is an option that controls the treatment of strobed events If the Create Separate Events for All Strobed Codes option in the Plexon Files tab of the Tools Options dialog box is unchecked all strobed events simply show up as events with a channel of 257 When the Create Separate Events for All Strobed Codes option is checked the strobed events are expanded into separate events for each possible data word value The strobed events are given a name corresponding to the value of their associated data word the format of which can be controlled by using the Name Generation Prefix and the Code Represen
223. he Adaptive Templates History view In the Templates vs Time view the template waveform amplitude is plotted versus the sample number within the waveform on the X and Y axis but the Z axis represents time elapsed in the file the start of the file is initially closest to the eyepoint and the end of the file is furthest away Each template waveform is rendered as a solid line as in the Waveforms view at the time when that template was put into effect and a solid sheet is rendered with the same sample by sample shape as the template waveform extending through time until the next template was put into effect or the file ends 162 Offline Sorter Offline Spike Sorting Software Spike Sorting Templates Vs Time In the screenshot above there were a total of six different templates used for this unit during the adaptive template sort of this channel Most of the adaptive templates were put into effect near the start of the file closer to the eyepoint with only a single template change occurring during the second half of the file away from the eyepoint The Templates Vs Time view shows the history of the templates for a single unit at a time Selecting a different unit either by clicking a unit in the Units view or using the Select Next Unit or Select Previous Unit menu items will change the display to show the history of template waveforms for that unit The Templates Vs Time view behaves identically to the 3D Clus
224. he files that match a template for processing The files are not actually processed until the Process command is encountered The template should include the full path to the files Syntax Dir template e g Dir c myfiles plx will queue all of the plx files in the c myfiles directory for pro cessing Note Itis not mandatory to specify any input data files using the File or Dir commands in a batch command file When a batch file that does not specify any input data files is executed from within Offline Sorter Software it will execute on the currently open data file 6 9 1 3 ForEachFile The ForEachFile command specifies an action to be done for each file being processed For some commands the action is performed before the channels are processed for others it is only performed after all channels have been processed See Notes below Syntax ForEachFile sub command e g ForEachFile ExportToNex A list of the sub commands for the ForEachFile command follows e Save saves the file The saved file name is the same as the input file name with a numeric suffix added on to the original filename same as File Save menu option 314 Offline Sorter Offline Spike Sorting Software Batch File Processing e ExportToPlx or SaveToPIx saves the file as a new plx file same as File Export to New PLX menu option possibly with a numeric suffix e ExportToP 2 or SaveToPI2 saves the file as a new pl2 file same as
225. he goal is to view the field potentials from the FP Source for the corresponding channel in the Timeline view One method that will work in many cases is just to double click on the FP Source in the Sources view or equivalently highlight the FP Source right click and select Select Corresponding Channel in Source from the context menu Spike 1 64 1 Cont 65 128 1 Cont 129 192 1 193 256 1 Raw Chs File AssociD Bits _MaxV_ NPW NPre Sorting 1 64 iY 16 2500 000032 8 11 32 1 64 Y 16 2500 0000 65 128 Y 16 2500 0000 129 192 Y 16 2500 0000 This causes Offline Sorter Software to make a reasonable guess as to which channel in the selected Source FP corresponds to the current SPK channel This method only works in cases where the two Sources have the same number of channels and so it is obvious which channel in the highlighted Source corresponds to the SPK channel The Timeline view is configured to show both the SPK channel and the corresponding FP channel which in this case happened to be Offline Sorter Channel 195 Spike Ch 3 SPKO3 Showing f Eont Ch 195 FPO3 Las Control Displays Center Selected Zoom 2 50 m Thr 6 61 165 pi Detect Waveforms Options Detect L2 Low cut Filter None 4 pole Cutoff Freq 250 Hz Apply Scroll Range 1 000 0 3259 Zoom Out Zoomin lt w 284 Offline Sorter Offline Spike Sorting Software Details of Channe
226. he mouse pointer is moved while pressing the left button the Waveforms view and Timeline view select the waveform currently closest to the mouse pointer On the bottom status bar of the application Offline Sorter Software displays the exact X Y and Z if applicable feature coordinates for the highlighted waveform x 606 9104 Y 2395 08 Z 2395 08 as well as the timestamp associated with the highlighted waveform 3 7 2 3 Controlling Which Waveforms Are Displayed If the Control Displays checkbox is selected the scroll bar at the bottom of the Timeline View can be used to control the temporal range of the waveforms shown in the Clusters views The Clusters views parameter in the Control Grid can be used to restrict which waveform types are displayed in the Clusters views either All or only those shown in the Waveforms view Unit clusters in feature space can be defined by using either the manual Contours method See Units View on page 42 or other sorting methods Specific waveform points from a unit cluster can be added or removed by selecting the 56 Offline Sorter Offline Spike Sorting Software 2D and 3D Clusters Views Units Add Remove Waveforms from the Selected Unit menu commands The Waveforms view can be used to add or remove waveforms by crossing them Also you can select and add or remove individual waveforms from a unit in the Continuous Data Display by using the right click menu 3 7 2 4
227. he spike The Offline Sorter System aligned extraction thresholding algorithm uses this approach Here is an example of the difference between standard threshold crossing top and aligned extraction bottom Note how the waveform bundles and the corresponding PCA clusters are more well defined when aligned extraction is used Version 4 0 311 6 Reference Topics Besides the improved timestamping accuracy and jitter reduction the more well defined clusters improve the performance of the automatic unit finding algorithm 6 8 Enhanced PCA Feature Space In addition to the standard PCA principal components analysis feature space the Offline Sorter System supports an improved version of PCA which was developed by Plexon specifically for spike sorting applications called Enhanced PCA Standard PCA determines a projection in which the first component is in the direction of maximum variance in the original data the second component is in the direction of second greatest variance and so on Enhanced PCA uses not only variance but separability of the data in calculating the projection For example if there is a sample position where a group of spikes has a large amount of amplitude variance but it is basically noise without any structure within the distribution of amplitudes at that sample time Enhanced PCA will de emphasize that component of the data in the resulting projection In contrast samples where there is a high
228. here _ _ Width width J Be The Signed Energy of the Signal is the Energy of the Signal multiplied by 1 when the average amplitude of the raw signal in the energy window is positive and multiplied by 1 when the average amplitude is negative The Signed Energy of the Signal enables the threshold to differentiate between energy due to large negative excursions and energy due to large positive excursions It is instructive to look closely at these calculated quantities for typical waveforms The top yellow trace is the calculated quantity and the bottom trace is the raw waveform amplitude Energy of the Signal Nonlinear Energy of the Signal 3 11 4 Stereotrode and Tetrode Waveform Detection Offline Sorter Software supports extracting stereotrode and tetrode waveforms from continuous data To begin configure the continuous channels as either stereotrode or tetrode channels From the Info Grid Views select the Channel Map view and configure the channels described in Section 6 2 Channel Mapping on page 286 When a stereotrode or tetrode channel loads the Waveform Detection view and the Timeline view show 2 or 4 traces instead of just one Observe this arrangement in the following illustration 78 Offline Sorter Offline Spike Sorting Software Waveform Detection View Peak Histogram ax 4 2D Clusters 3D 3D Clusters Surface Peak Histogram Control Displays Center Selected Zoom 1 0 5
229. how 3 Sigma Peak Heights Line If checked shows blue dotted horizontal lines that represent standard deviations of noise The noise is calculated by setting the Threshold to 3 standard deviations of the original signal and then removing waveform segments 250 msec before and 750 msec after the threshold crossings e Show Dots at Sample Locations When showing spike or continuous data the Timeline view connects the sample points with lines When this option is checked a point is drawn at the time voltage coordinates of each spike or continuous data sample When the channel Data Type is Stereotrode or Tetrode the display divides up to vertically stack 2 or 4 continuous traces or spike waveforms as shown in the Section 6 3 Stereotrode and Tetrode Data on page 291 For both the waveform and continuous data displays the status bar on the bottom of the screen shows the time and the voltage corresponding to the current mouse position within the display Free Mem 190 510M Dropped None t 0 11 Y 156 46 my x 3 7 2D and 3D Clusters Views Although they are treated as separate views in Offline Sorter Software the 2D Clusters view and the 3D Clusters view share many features in common Both Clusters views show the waveforms in feature space The 2D Clusters view shows the waveforms as points in a 2 dimensional feature space and the 3D Clusters view displays points in a 3 dimensional feature space Both the 2D and 3D Clusters views
230. how a Den sity Histogram 2D Clusters 40x 2D Clusters 303D Clusters Surface Peak Histo 62 Offline Sorter Offline Spike Sorting Software 2D and 3D Clusters Views The density histogram can be displayed in linear or log scale When the density histogram appears Offline Sorter Software enables the Density Histogram Settings item on the right click menu Select this item to display a floating dialog box that can be used to set the contrast enable disable smoothing control the bin size and to choose between linear or log scaling for the density histogram color assignments The Number of Histogram Bins parameter controls the number of bins per side that make up the density histogram From the Smoothing drop down list an algorithm can be selected to smooth out the density histogram by combining and averaging the counts in adjacent bins The Histogram Count for Saturation parameter controls the mapping between the dot density and the grey level scale If Histogram Count for Saturation 10 for instance then all cells with more than 10 dots appear in white 2D Clusters ee Tilt Number of Histogram Bins 100 Histogram Count for Saturation 100000 Contrast Smoothing Gaussian v Log Scale T 2D Clusters 3D 3D Clusters Surface Peak Histogram Both the dots and the density histogram can be viewed simultaneously in the 2D Clusters view by selecting the Show Dots and Density H
231. hreshold line on the histogram in the Waveform Detection view to the left or right can set the threshold Click Detect to extract the waveforms using the specified detector total waveform length prethreshold period dead time and threshold level You can automatically set a threshold based on the statistical properties of the peak heights and perform waveform detection on all channels as opposed to doing a single channel at a time For complete information see Section 5 5 14 Waveforms Detect on page 249 4 3 Waveform Acquisition From Spike Waveform Data 4 3 1 Opening the Spike Waveform Data File Offline Sorter Software can read native data files containing spike timestamps and spike waveform segments created by the Plexon MAP system PLX or OmniPlex System PLX or PL2 as well as other data acquisition systems CyberKinetics CED Spike 2 DataWave MultiChannel Systems Panasonic MED Neuralynx etc 124 Offline Sorter Offline Spike Sorting Software Waveform Acquisition From Spike Waveform Data Offline Sorter Software can also open multiple Plexon PL2 files and sort and analyze all of them together See File Open Multiple Files on page 226 To open a data file Click the File Open toolbar button select File Open menu command Or To open multiple PL2 files Select File Open Multiple Files menu command To open a spike waveform file from another vendor s hardware Selec
232. id or x i e Pressing the button in the toolbar 5 5 4 Waveforms Create Time Segments As mentioned in Section 3 2 5 Time Segments View on page 34 Offline Sorter Software can divide up the time span covered by the experiment into an arbitrary number of time segments By default Offline Sorter Software will divide up the file into 10 evenly spaced time segments To change how the file is divided into time segments select Waveforms Create Time Segments from the main menu or Create New Segments from the right click menu in the Time Segments view A dialog will appear that allows one to divide up the file into time segments in various ways Version 4 0 243 5 Menu Options Create New Time Segments The data file covers a time duration of 29 481725 secs D Create Segments with a specified duration Time duration for each Segment 60 secs If the last Segmentis shorter than 30 Secs combine it with the previous Segment D Create a single Segment covering the time span From 0 secs Name To 29 481725 secs _ Create multiple arbitrary Segments covering the time spans From 9 secs Name To 29 481725 secs Add Segment me cena oo To divide up the file into time segments that have a specified duration select the first radio button With this method there may be leftover time at the end of the file the method allows one to specify whether that leftover time sho
233. in Section 4 5 12 Parameter Scans on page 179 e Set As Top Row After highlighting several rows will set the Show and Display Color values for all highlighted rows to be that of the first highlighted row e Randomize Colors Picks random Display Colors 3 2 6 Sort Details View The Sort Details view shows information about the channels that have been sorted during this session of Offline Sorter Software Channels that have not yet Version 4 0 35 3 Views and Displays been loaded during this session of Offline Sorter Software will have s in their entries O o gt amp S a 62225 8 to 97007 7 3 NEINEINEENEEINEE NEN NED i i i i i i i i i MVENVENENENENENE INE MVENVENENENENENE NEN The X Y and Z columns show which feature is selected for each dimension of feature space The R columns show the range covered by the feature values the column width can be expanded by dragging the column header to see these values The contents of this view is essentially what is written out to a TPL file For more information see Section 4 8 4 1 Saving Templates on page 212 3 2 7 Events View The Events view displays the events that are present in the data file Individual events can be selected to show in the Timeline and Rasters views For more information see Section 4 4 3 Examining Events on page 137 3 2 8 Stats View The Stats view contains the results
234. in groups of four Data Type automatically selects the appropriate mode for Plexon PLX PL2 Datawave UFF and Neuralynx files For more information see Section 6 3 Stereotrode and Tetrode Data on page 291 Sort Method is the cluster selection method The following sections provide procedures for the available selection methods e Templates Section 4 5 4 Semi Automatic Clustering using Templates on page 157 38 Offline Sorter Offline Spike Sorting Software Control Grid e Boxes See Section 4 5 5 Manual Clustering using the Boxes Method on page 163 e Lines Section 4 5 6 Manual Clustering using the Lines Method on page 165 e Bands Section 4 5 7 Manual Clustering using the Bands Method on page 168 e K Means Section 4 5 8 Semi Automatic Clustering using K Means on page 171 e Standard E M Section 4 5 9 Semi Automatic Clustering using Standard E M on page 175 e WValley Seeking Section 4 5 10 Automatic Clustering using Valley Seeking Method on page 176 e T Distribution E M Section 4 5 11 Automatic Clustering using T Distribution E M Method on page 178 e Parameter Scans Section 4 5 12 Parameter Scans on page 179 e Scanning Methods See Section 4 5 13 Automatic Clustering Using the Scanning Methods on page 182 When one of these options is selected the current sorting mode is set but a sorting operation is not actually initiated Zoom
235. ince the Set commands to change all possible parameters do not exist in the batch syntax This can lead to non reproducible results so it is good policy to do explicit Sets of all parameters that can affect the results in the batch file or use the Set OptionsFromFile batch command to get all parameters into a known state All processing done by batch commands is done in a certain order for each of the files being processed Each of the following steps may or may not be specified in the batch commands and some commands may not be relevant for a given channel The order is 1 Apply a channel map 2 Invalidate artifacts 3 Apply a tpl file 4 Process each channel The order of processing for each channel is a Apply upsampling to continuous channels b Apply filtering to continuous channels c Detect waveforms for continuous channels d Invalidate waveforms the KeepWf and InvalidateWf commands 322 Offline Sorter Offline Spike Sorting Software Batch File Processing e Perform alignment f Delete all units g Perform sorting h Remove outliers i Remove short ISI waveforms j Assign unsorted waveforms to clusters k Dump sort summary to PowerPoint Log stats m Perform parameter scans 5 Invalidate artifacts if the InvalidateArtifacesAfter command is used 6 All saving and export to file operations e When importing a file from batch Offline Sorter Software by default uses the loader implied by the file extens
236. ing units See Statistical Principals in Experimental Design 2nd Edition by B J Winer page 232 The conversion of the F statistic to a p value is done via a calculation of the incomplete beta function See Numerical Recipes in C section 6 2 assuming a single tailed probability distribution Including or not including the unsorted units in the ANOVA calculation The Include unsorted waveforms as a unit in the statistics calculations checkbox in the Stats tab in the Tools Options dialog box controls whether the unsorted waveforms are treated as a unit in the statistics calculation or simply ignored The text in parenthesis on the Multivariate ANOVA line indicates the current setting for this option For additional information see Section 6 6 1 1 Some details on the MANOVA calculation on page 307 Note The multivariate ANOVA test requires a minimum of 2 clusters one of which can be the unsorted waveforms if they are included as a unit Also it does Version 4 0 307 6 Reference Topics not make sense to run the ANOVA test if any unit is defined that contains zero waveforms again including the unsorted waveforms if they are included Interpretation of the Multivariate ANOVA test This section includes information on the interpretation of the Multivariate ANOVA tests The test results appear in the lower half of the Stats view Assumption The distributions of points is normally distributed in 2D or
237. ing the Contours Method The Contours method of sorting is always available no matter which Sort Method is selected in the Control Grid This is different from pre 3 0 versions of Offline Sorter Software Pressing the Add Unit button on the toolbar of the 2D or 3D Clusters views will initiate Contour sorting Holding down the Control key while clicking and drawing in these views is also a shortcut to creating a new unit using the Contours method For details on how to use the Clusters views see Section 3 7 2D and 3D Clusters Views on page 52 146 Offline Sorter Offline Spike Sorting Software Spike Sorting If the 3D Clusters view is being used orient the eyepoint as described in Section 3 7 2 13 3D Clusters View Details on page 65 so as to maximally separate the clusters Version 4 0 147 4 Spike Sorting Step by Step 4 5 2 1 Adding a Unit The purpose of adding a unit is to identify a subset of waveforms that are presumably generated by the same neuron To add a unit using the Contour sorting method 1 In the Clusters view click Add Unit or hold down the ALT key and right click in the Clusters view and select the Add Unit menu command or hold down the CTRL key 2 Move the mouse pointer to the Clusters view The pointer changes to a drawing hand 148 Offline Sorter Offline Spike Sorting Software Spike Sorting 3 Click the left mouse button and drag a line around th
238. inished moving or resizing a box the waveforms are sorted according to the new box positions Note The Boxes sorting algorithm is greedy in that a waveform is sorted into the first unit starting with Unit a for which it passes through both boxes If a waveform passes through both boxes for several units it is assigned the lowest lettered unit 4 5 6 Manual Clustering using the Lines Method Select the Lines method of sorting by selecting Lines in the Sort Method droplist in the Control Grid or by selecting the Sort Change Sort Method Use Lines menu item The Lines algorithm works in waveform space not in feature space The goal of the algorithm is to define a list of lines in time voltage space for each unit To be sorted into a unit the waveform must intersect all of the lines defined for that unit To add a unit using the Lines sorting method perform the following steps Version 4 0 165 4 Spike Sorting Step by Step 1 Press the Add Unit button in the Waveforms view Invalidate Wfs E or right click in the Waveforms view and select the Add Unit menu command The cursor turns into a drawing hand 2 Define the first line in the unit definition by crossing a bundle of waveforms exactly as described in Section 4 5 3 Manual Clustering using the Waveform Crossing Method on page 153 As soon as the mouse button is released after a bundle of waveforms is crossed all waveforms that intersect the
239. inuous Data e Channels indicates the total number of continuous channels in the file e Peak Voltage at ADC input mV shows the voltage at the ADC input that will produce the maximum analog to digital converter count for continuous channels e A D Resolution bits is the resolution of the analog to digital converter for the channels Events e Channels indicates the number of events with unique ID codes in the file Total Count indicates the total number of events in the file for all event channels Note Some items listed under Waveforms e g Data Type Waveform Length can actually be different per Source for some file formats The values for either the first or the currently selected Source are shown in this case 3 2 5 Time Segments View Offline Sorter Software has the ability to divide a file into an arbitrary number of time segments These time segments can represent periods when different experimental conditions were in effect or they can just be evenly spaced within the file in order to see how conditions evolved as the experiment progressed Offline Sorter Software can calculate sort quality parameters on a segment by segment basis and can display the time segment boundaries on various views The Time Segments view shows the current division of the file into time segments in a tabular format with a count of waveforms and the values of calculated sort quality measures for each time segment Time segme
240. inuous data 6 11 Large File Tips The following are some settings and usage tips that can significantly speed up Offline Sorter Software when working with large PLX or PL2 files 6 11 1 Prefer PL2 files In general PL2 files will load into Offline Sorter Software much faster than will PLX files or data files from other vendors Version 4 0 325 6 Reference Topics 6 11 2 Statistics Calculations In general Offline Sorter Software must recalculate sort quality statistics whenever any change is made to the sorting on a channel This can take significant time especially if unit pair wise statistics are being calculated The Stats tab of the Tools Options panel contains checkboxes that enable or disable the calculation of the advanced sort quality statistics and also the unit pair wise Statistics If the statistics are not of interest uncheck these items to speed up sorting operations 6 11 3 Buffer Sizes On the General tab of the Data Import Options page there are two Buffer Size settings to be aware of In both cases these buffer sizes are specified in megabytes These values should always be less than the amount of RAM in the computer or severe performance degradation can occur The Reading Buffer Size for data files is the size of the temporary cache Offline Sorter Software uses when reading data from files Increasing this number generally speeds up reading larger files but might slow down the reading of smaller fil
241. ion e g the Cyberkinetics loader uses nev files If the file to be loaded does not have a recognized extension Offline Sorter Software uses the loader corresponding to the currently selected default File Type You can use the Set DefaultFileType command to set the default File Type e Numerical values to use in the Set FeatureXXX commands OPC 1 1PC2 2PC3 3 Slice 1 4 Slice 2 5 Peak 6 Valley 7 Peak Valley 8 Energy 9 Nonlinear Energy 10 Timestamp 11 PC 4 12PC5 13 PC 6 14PC7 15 PC 8 Version 4 0 323 6 Reference Topics 16 Peak FWHM 17 Valley FWHM 18 Peak Tick 19 Valley Tick 20 Area under Waveform 21 Slice 3 22 Slice 4 23 Peak to Valley Ticks 24 Valley to Peak Ticks 25 Sart Energy 26 Previous ISI 27 Next ISI 6 10 Sample Batch Command Files Offline Sorter Software ships with some sample batch command files which are installed in the SampleBatchFiles subdirectory of the main Offline Sorter installation directory The sample batch command files do not contain any File or Dir commands to specify the input data files Thus they all make use of the feature described above where they will operate on the currently loaded data file when they are executed from within Offline Sorter Software The general procedure for running the sample batch command files is as follows 1 Load a data file using File Open or File Import If there are not any data files available sample data files can be downloa
242. ion has an ofi extension Then the same Import Parameters can be restored by loading the file using the Load from ofi File button If Load Using Companion ofi File is clicked Offline Sorter Software looks for an ofi file with the same base name the filename minus the extension and loads the binary file using the Import Parameters in the companion ofi file The format of the OFI file is a number of lines in the format Parameter Value The parameters correspond to the edit boxes in the above dialog with the keywords NChan Offset Freq MaxMV ResolutionBits and Swap respectively For example a typical OFI file could be e NChan 81 e Offset 0 e Freq 25000 000000 e MaxMV 10 000000 e ResolutionBits 16 e Swap 1 Note Explicit support for loading Plexon ddt files is no longer present in Offline Sorter Software However ddt files can be imported with the Generic Binary importer using the Data Offset and other parameters shown in the above screenshot Version 4 0 225 5 Menu Options 5 1 2 13 Generic Neuroshare Import In addition to the known vendor formats that can be imported via a vendor supplied Neuroshare DLL Offline Sorter Software provides a mechanism to use any arbitrary Neuroshare DLL to load a data file To do this first open the Tools Data Import Options dialog box and click the Neuroshare tab and specify the location of the Neuroshare DLL on disk in the Ge
243. ion of time The time range viewed can be expanded or reduced and it can be repositioned within the file The display can be used to specify digital filtering select the threshold for waveform extraction and select individual waveforms for inspection See Section 3 6 Timeline View on page 48 2D Cluster View The 2D Clusters view shows the waveforms as points in 2D feature space Each axis can be selected from any of the Section 6 4 Features Available for Sorting on page 294 This display can be used to select the clusters of waveforms For more information see Section 3 7 2D and 3D Clusters Views on page 52 3D Clusters View The 3D Clusters view shows the waveforms as points in 3D feature space Each axis can be selected from any of the Section 6 4 Features Available for Sorting on page 294 This display can be used to select the clusters of waveforms For more information see Section 3 7 2D and 3D Clusters Views on page 52 Surface View The Surface view shows the density of points in 2D feature space plotted as a three dimensional surface plot See Section 3 8 Surface View on page 69 Clusters vs Time View The Clusters vs Time view shows the points in 2D feature space plotted against time on the third axis This is useful to see how the waveforms changed as a function of time See Section 3 9 Clusters vs Time View on page 71 Templates vs Time View The Templates vs Time view is only relevant if A
244. ions appear References N Schmitzer Torbert J Jackson D Henze K Harris A D Redish Quantitative Mesaures of Cluster Quality for use in Extracellular Recordings Neuroscience 131 2005 1 11 6 7 Thresholding Algorithm Aligned Extraction By default the Offline Sorter System uses a standard thresholding spike extraction algorithm which defines the time at which the spike waveform crosses exceeds the threshold as its timestamp While this is a classic method of thresholding it has some drawbacks First it means that as the threshold is raised or lowered the threshold hits the spike at different points on a rising or falling edge potentially resulting in the timestamp changing by one two or more sample positions Another manifestation of the same effect is that spikes of 310 Offline Sorter Offline Spike Sorting Software Thresholding Algorithm Aligned Extraction different amplitudes will have slight timestamp offsets relative to each other While the size of these timestamp errors or jitter is too small to cause problems in most spike train analyses it does have a significant effect on spike sorting it causes smearing of clusters in feature space The basic problem with threshold timestamping is that it does not use an unambiguous feature in time voltage space of the spike as the reference to which a timestamp is attached The obvious choice for such a reference is the largest peak within t
245. ions within Offline Sorter Software including setting options Window Run Help Recalculate the Principal Components Align Waveforms Adjust Gains Invert Waveforms Re Threshold Waveforms Interval Selection Remove Short IS Waveforms Remove Outliers Assign Unsorted to Closest Unit Invalidate Cross Channel Artifacts Invalidate High Amplitude Artifacts Density Leveling All Options wef Options OPT q Data Import Options 5 8 1 Tools Recalculate the Principal Components To recalculate the principal components using the time range between the Sort Start and Sort End time points select the Tools Recalculate Principal 262 Offline Sorter Offline Spike Sorting Software Tools Menu Components menu command and recalculate using All the points and Only the points specified by sort start and sort end Recalculate Principal Components Recalculate Principal Components Using Use Enhanced PCA Waveforms To Use All the waveforms All valid unsorted waveforms Use only waveforms within the time range Maximum waveforms to use 10000 Waveform Points To Use All the points Only the points between sort start and sort end Do not show this dialog again mre 5 8 2 Tools Align Waveforms This menu item is the same as the Waveforms Align option Select this menu item to align the spikes in a spike channel in time For additional de
246. is an option to Show or Hide the waveforms marked as invalid in the Waveforms and Clusters views Offline Sorter Software retains this value between invocations Electrode Enable is only relevant if stereotrode or tetrode data is being processed by Offline Sorter Software Then this droplist will show checkboxes for each of the electrodes within the stereotrode or tetrode Unchecking the checkboxes will cause data from that electrode to be ignored when calculating PCA and feature values 3 4 Units View Offline Sorter Software provides information about each of the currently defined units in the Units view along with a display for the waveforms that are currently unsorted The top row of graphs of this display shows the template average waveform for the unit solid line as well as 3 standard deviations from the template dashed lines The edit controls can be used with the spin buttons to adjust the Fit Tolerances for Template Sorting The default template Fit Tolerance 70 in the preceding screenshot can be set by using the Templates tab on the Tools Options menu panel After a new value for the Fit Tolerance is set you can elect to apply the new value to existing units or to only use the new value when new units are defined For additional information on templates see Section 6 5 2 Template Algorithm on page 300 The bottom row of graphs of this display shows the Interspike Interval Histograms The X axis of each his
247. is the vertical zoom factor in the Waveform View and the Timeline View The up or down arrows can be used to increase or decrease the factor by steps but any floating point value can be entered by typing the numbers By default the up down arrows increase or decrease the factor by increments of 0 1 when the current zoom factor is small and by increments of 1 0 when the zoom factor is higher The value at which the 1 0 increment applies can be adjusted by typing the value in the Zoom control steps by 1 0 starting at value box on the Display tab in the Tools Options window Max w f draw Maximum waveform draw is the maximum number of waveforms drawn in the Waveform View and the Clusters Views For example if there are 30000 waveforms in the active channel and Max w f draw is set to 10000 then only every 3rd waveform is drawn Change the Max w f draw setting from the large default value only to improve drawing speed on slower computers when viewing channels with many waveforms When the number of waveforms in a channel is greater than the Max w f draw setting the Waveform View shows the waveform count section highlighted in red and there is a Version 4 0 39 3 Views and Displays AN divided by indicator that shows the fraction of waveforms that are being displayed e g for the preceding example the divided by indicator is 3 CAUTION Misleading Sort It can be misleading to sort units on a channel where Max w f draw is les
248. isplay hold down the SHIFT key then hold down the left mouse button and move the mouse in the direction that to pan the display The pointer becomes a hand shaped symbol when zooming or panning occurs Note The displays can be zoomed and panned using toolbar buttons or context menu entries Both zooming and panning can be reset by using the Reset Zoom toolbar button or by using the Reset Range and Zoom context menu option This sets the display to default such that all the points are visible in the field of view 3 7 2 11 Context Menus Both of the Clusters views support context menus commonly known as right click menus although the menu items available in each differ slightly To bring up the context menu hold down the ALT key and then click the right mouse button The menu items common to both the 2D and 3D Clusters views are e Add Unit creates a new unit using Contour sorting i e circling the waveforms in the new unit This is the same as pressing the Add Unit button in the Per View Toolbar e Find Units executes the currently selected Sort Method to find new units This is the same as pressing the Find Units button in the Per View Toolbar e Clear Centers this command is only relevant if the K Means or Std E M Sort Methods are in effect and some unit centers have been selected on the view See Section 4 5 8 Semi Automatic Clustering using K Means on page 171 and Section 4 5 9 Semi Automatic Clustering using S
249. istograms right click shortcut menu Version 4 0 63 3 Views and Displays Showing 1D Histograms The 2D Clusters view can display histograms of the point distributions in each dimension by selecting the Show 1D Histograms right click menu command The histograms are displayed along the margins Showing Ellipses The 2D Clusters view can display standard deviation ellipses around clusters by selecting the Show Ellipses right click menu command or by pressing the E key while the 2D Clusters view has input focus The ellipses are oriented along the principal axes of the cluster of points with the lengths of the major and minor axes for the ellipse being the standard deviation sigma of the points along each axis scaled by the value of the Outlier Threshold in the Control Panel The ellipses in the following illustration show the 4 sigma boundaries around each unit Offline Sorter Software draws the ellipses with dashed lines in the color associated with the unit 64 Offline Sorter Offline Spike Sorting Software 2D and 3D Clusters Views Region Zooming The 2D Clusters view can be zoomed in to an area of interest by clicking dragging a box using the middle mouse button Like the other methods of zooming or panning the display this adjusts the X and Y display range 3 7 2 13 3D Clusters View Details The 3D Clusters view allows changing the eyepoint in 3D feature space via mouse and keyboard actions in order to
250. it e either in the Waveforms view e See Adding Waveforms to Selected Units in the Waveforms View on page 154 and e Section 4 6 3 Cleaning the Selection on page 187 e or in the Clusters views Version 4 0 177 4 Spike Sorting Step by Step e See Adding Waveforms to Selected Units in the Clusters Views on page 150 and e See Removing Waveforms from Selected Units In the Clusters Views on page 151 4 5 11 Automatic Clustering using T Distribution E M Method Select the T Distribution E M method of sorting by selecting T Dist E M in the Sort Method drop list in the Control Grid or by selecting the Sort Change Sort Method Use T Distribution E M Sorting Method menu item The T Distribution E M algorithm is an iterative procedure that operates on the points in feature space to assign the waveforms to an optimal number of clusters For more details see Section 6 5 8 T Distribution E M Algorithm on page 305 The T Distribution E M algorithm operates differently depending on which view initiates it and what options are set see Section 6 5 1 Feature Space Used for Sorting on page 299 To run the T Distribution E M clustering algorithm Click Find Units in the toolbar or select Find Units from the right click menu or select Sort Sort Current Channel Run T Dist E M Sorting If it is not cancelled the algorithm run until convergence or until a specified maximum number of iterations compl
251. it in the Control Grid then only the waveforms from the selected Unit are shown if they are set to All then all waveforms are shown 242 Offline Sorter Offline Spike Sorting Software Waveforms Menu 5 5 2 Waveforms View Only Unsorted Waveforms This is a toggle able menu item When it is selected checked the Waveform view and optionally the Clusters views and the Timeline view will show only the waveforms that have not yet been sorted they belong to the Unsorted unit Selecting this menu item is equivalent to either of the following e Selecting Unsorted from the Waveform View droplist in the Control Grid or e Pressing the button in the toolbar Whether this selection applies only to the Waveform view or to the Clusters views and the Timeline view is determined by the setting of the Cluster Views and Timeline View droplists in the Control Grid if they are set to As Above meaning the setting for the Waveform View above it in the Control Grid then only the waveforms from the selected Unit are shown if they are set to All then all waveforms are shown 5 5 3 Waveforms View Only Selected Time Segments This is a toggle able menu item When it is selected checked the Waveforms view and the Clusters views will show only the waveforms that are within the currently selected Time Segment Selecting this menu item is equivalent to either of the following e Checking the Only Segment checkbox in the Control Gr
252. item Also there is a toolbar that includes icons for these functions but this toolbar is not displayed by default To display this toolbar select Toolbars tab under Windows Customize and click Animation in the Toolbars list You can increase the usefulness of the animation features by configuring the Waveform and Clusters views to only show the waveforms within the current time range thus what they display changes while the animation is in progress You can accomplish this with the Control Displays checkbox in the Timeline view If these checkboxes are checked the Clusters views or the Waveforms view or both show only those waveforms that appear in the Timeline view You Version 4 0 239 5 Menu Options can use the Zoom In and Zoom Out buttons on the Timeline view to control how many waveforms are visible in each frame of the animation Several commercially available screen capture packages are capable of making MPEG movie files of any on screen activity including the Offline Sorter displays 5 4 Select Menu The Select menu contains entries for navigating channels units and waveforms It also contains operations to select feature spaces used for Sorting Select Waveforms Sort Units Next Channel Previous Channel Next Unit Previous Unit Next Waveform Previous Waveform Next Segment Q2ARA3R BY Previous Segment Feature Space Definitions gt 5 4 1 Select Next Channel
253. ith each waveform To do so click the Append Raw Waveform A D Data checkbox The A D values appear in additional data columns after the exported feature columns To export only the waveform A D values deselect all features from the Chosen list box and click the Append Raw Waveform A D Data checkbox The number of appended columns is equal to the number of points per waveform The waveform A D values can be exported in units of microvolts the default or in raw ADC counts by clicking the radio buttons near the checkbox Select What File s to Store the Exported Data in by typing a filename or selecting one using the Browse button For exports involving multiple 208 Offline Sorter Offline Spike Sorting Software Saving the Sorting Results channels into multiple files Offline Sorter Software appends a channel number to the filename entered 9 Click the Export button 10 For exports to Excel save the spreadsheet using Microsoft Excel 4 8 3 2 Exporting Per Channel Data Selecting the File Export Per Channel Data menu selection displays the following window Delimiter to use between values Matlab file O Excel 7 Export a Row of Column Headers Comma F What to Export for Each Channel Available Channel Raw Channel Name Channel Name Raw Add gt gt Number of Unsorted Waveforms Sort Method Used p 2D Add All F 2D F Hypoth D F 2D lt lt Remove F Error D F 2D i J
254. its Remove Waveforms from the Selected Unit menu command 3 Move the mouse pointer into the Waveforms view ey The pointer changes to a drawing hand i 4 Click the left mouse button and drag a line across the waveforms to remove The crossed waveforms are removed from the selected unit For methods to display only the selected unit while removing waveforms see Section 4 6 3 Cleaning the Selection on page 187 Deleting Units Use the following procedure to delete a unit To Delete a Unit 1 Select the unit by clicking it s template in the Units view 156 Offline Sorter Offline Spike Sorting Software Spike Sorting 2 Click the Delete Unit button in the toolbar HA x pos Delete Unit Add Wfs Remove Wfs Invalidate Wfs The selected unit is deleted To delete all the sorted units Select the Units Delete All Units menu command or hold down the ALT key and right click in the Clusters view and select the Delete All Units menu command 4 5 4 Semi Automatic Clustering using Templates Select the Templates method of sorting by selecting Templates in the Sort Method drop list in the Control Grid or by selecting the Sort Change Sort Method Use Templates From Designated Waveforms menu item The Templates algorithm works in waveform space not in feature space The goal of the algorithm is to select an existing waveform to serve as the template waveform for a new unit However the Clusters
255. ke Source associated with the channel the continuous Source associated with the channel or both 6 1 4 Viewing Corresponding Channels As explained above when Offline Sorter Software first loads a data file it generally treats all Sources and the Offline Sorter Channels as being independent and unrelated to each other An operation like waveform detection creates a new spike Source that is related via the AssocID to a continuous Source And the Offline Sorter Channels that have had waveform detection performed on them will refer to both the original continuous Source and the new spike Source Loading that Offline Sorter Channel will thus display both the continuous data from the continuous Source and the spikes from the spike Source together in the Timeline view However even if waveform detection was not done within Offline Sorter Software there can still be real world relationships between spike and continuous Sources In the example shown in the above screenshots there is a straightforward correspondence between each raw channel in the SPK Source with the same numbered channel in the continuous WB Source as the signal for both came from the same physical electrode In this example the same correspondence holds also between the SPKC and FP Sources there were only 64 total physical electrodes in the experiment each physical electrode contributed a SPK channel a raw wideband WB channel a low cut filtered SPKC channel
256. ke sorting techniques see Wheeler and Heetderks 1982 Schmidt 1984 Lewicki 1998 Wheeler 1999 For many data acquisition systems spike detection is done on line and Offline Sorter Software can read in and use the already detected spike waveforms For continuously recorded data files Offline Sorter Software can perform spike detection by using a threshold crossing algorithm When spike waveforms are present Offline Sorter Software helps to separate the waveforms collected from single or multiple electrodes into distinct waveform groups or classes that are associated with individual neurons Each class of waveforms is called a unit or a cluster Offline Sorter Software currently provides the following spike sorting algorithms grouped into categories e Manual Methods e Waveform Crossing e Contours e Boxes e Lines e Bands e Semi automatic Methods e Templates e K Means e Standard E M Expectation Maximization 10 Offline Sorter Offline Spike Sorting Software Sorting Techniques e Automatic Methods e Valley Seeking e T Distribution E M e Scanning methods All sorting methods work in so called feature space except for the Waveform Crossing Template Boxes Lines and Bands sorting methods Instead of using the entire raw waveform sorting methods that work in feature space characterize the essence of the waveform by using a few calculated features The feature space can be defined by selecting from among se
257. key and left clicking on a unit in the Units view selects the clicked unit but leaves the previously selected unit s in the multiply selected state denoted by a dashed box drawn around the unit Holding down the SHIFT key and left clicking a unit selects that unit and keeps the previously selected unit s in the multiple select state and also multiple selects all units between the previous and the clicked unit If Sel Wave is selected from the Waveform View drop list Offline Sorter Software displays only the waveform currently selected in the Timeline view or the Clusters view If Unsorted is selected from the Waveform View drop list Offline Sorter Software displays only the unsorted waveforms regardless of what unit is selected This selection can help to add unsorted waveforms into an existing unit you can view only unsorted waveforms yet still have the target unit selected If this selection is chosen use one of the following waveform selection options Use Only Visible Waveforms or Use Only Unsorted Waveforms For more information see Section 4 6 2 Waveform Selection Options on page 186 Version 4 0 185 4 Spike Sorting Step by Step Controlling waveforms in the Clusters Views by Units By default the type of waveforms shown in the Clusters views matches those shown in the Waveforms view because the Clusters Views parameter in the Control Grid defaults to As above Draw Grid Refr ISI us 1000
258. l Select either Spline or Linear from the Interpolation Method droplist The possible choices for the up sampled frequency appear in the New Frequency droplist The data is immediately up sampled as the Up Sampling frequency is selected Note that it may not be apparent that the up sampling was performed on the continuous data by looking at the Timeline view It is instructive to select the Show Dots at Sample Locations for Continuous Data option in the right click menu of the Timeline view then changing the Frequency droplist The number of dots will increase as new sample locations appear between the previous ones 114 Offline Sorter Offline Spike Sorting Software Waveform Acquisition From Continuously Recorded Data 4 2 4 Adjusting Gains Offline Sorter Software utilizes the following model for channel gains e Spike channels can have a single Spike Preamp Gain for all channels and also a channel specific Spike Gain So the total gain for the spike signal path for a channel is the product of these gains e Continuous channels can have both a per channel Continuous Preamp Gain and a per channel Continuous Gain So the total gain for the continuous signal path for a channel is the product of these gains e Any given channel can have both spike and continuous data present Note that some file formats including PL2 files do not record preamp gains they only record total gains In these cases the pream
259. l File Select the name of the output file Click the Browse button to bring up a file browser Sources to Export only the Sources that are checked in the table are exported to the output PL2 file By default all Sources are checked Export Invalidated Waveforms If this item is selected Offline Sorter Software still writes waveforms that have been invalidated to the output PL2 file If unselected default Offline Sorter Software does not write the invali dated waveforms to the file Export Unsorted Waveforms If this item is selected the default Offline Sorter Software writes out the waveforms that are not sorted If unselected Offline Sorter Software writes out only sorted waveforms 5 1 9 File Export to NEX The main result of the sorting process is a set of spike trains arrays of timestamps corresponding to the sorted units You can export spike train data in NEX files for analysis with NeuroExplorer which is a software package that is separately available from Plexon See the Plexon website http www plexon com for additional information about NeuroExplorer Version 4 0 233 5 Menu Options To save the timestamps and other data such as continuous analog variables ina NEX data file 1 Select File Export to NEX The Export To NEX Options dialog box appears F Export To NEX Options File AA MEEN ACSA ESEE m EN BENESI What to Export _ Export Unso
260. l the window will be pinned and Version 4 0 Appendix A Navigating the Plexon UI remain open after you move the mouse away from the sidebar tab as shown on the right The Auto Hide button will also have changed direction and be pointed downward Note also that the File Info sidebar tab is gone on the right illustration _ Avg Units per Active Chan C Program Files Plexo Ee C Program Files Plexo Thursday March 03 2 640 by 480 30 000299 1789 Yes C Program Files Plexo Thursday March 03 2 1718000 59 575 105 32 Single 40000 000 40000 000 9724 4 5000 12 0 0 C Program Files Plexo C Program Files Plexo Thursday March 03 2 640 by 480 30 000299 1789 Yes C Program Files Plexo Thursday March 03 2 1718000 59 575 105 32 Single 40000 000 40000 000 9724 4 5000 12 Offline Sorter Offline Spike Sorting Software A 3 Standard Menu Items and Dialogs In Plexon software applications some menu items may have the same functionality across several applications These items are standard menu items and consist of the Window menu the Run menu and the Help menu A 3 1 Window Menu The Window menu contains three items Theme Layout and Customize e Theme Clicking on Theme displays a menu as shown below A theme is a color scheme that is part of the look and feel of the user interface Window Laut gt Plexon Blue Vista Customi
261. l by channel information such as thresholds gains and filtering See Section 3 2 3 Channel Parameters View on page 30 The File Summary view contains the global waveform and sorting parameters and file information See Section 3 2 4 File Summary View on page 32 The Time Segments view shows how the file has been subdivided into time segments See Section 3 2 5 Time Segments View on page 34 The Sort Details view shows the details about the sorting performed on each channel like what features and sorting methods were used See Section 3 2 6 Sort Details View on page 35 The Events view lists the external events present in the data file It allows events to be displayed with custom colors on the Timeline view See Section 3 2 7 Events View on page 36 The Stats view contains multivariate ANOVA tests on the separation of unit clusters and other sorting quality related statistical information See Section 3 2 8 Stats View on page 36 The PCA view displays the eigenvectors and values of the principal component analysis on the waveforms that is used to generate the principal component displays See Section 3 2 9 PCA View on page 36 The Scan view is an exploration tool that can automatically resort a channel numerous times while stepping the values of parameters This allows one to get a feeling for the optimal values of the sorting parameters See Section 3 2 10 Scan View on page 36 The Templates
262. l the individual PL2 files listed in the OMF being loaded into Offline Sorter Software The multiple file support in Offline Sorter Software relies on the existing Time Segments functionality each loaded file gets a Time Segment that covers the entire file This is useful for seeing where files begin and end 226 Offline Sorter Offline Spike Sorting Software File Menu To analyze multiple PL2 files 1 Select File Open Multiple Files from the main menu A file selection dialog appears By default this dialog is configured so that multiple PL2 files can be selected for the multiple file analysis session Or if a multiple file session has already been defined and saved to an OMF file the OMF file can be selected by first changing the Files of type droplist to show Multi File files omf G a Look in pl2spittest Name Date modified di TestSave 12 14 2013 9 28 AM File folder Recent Places all data 64 channels_sp _001 omf 12 2 20139 34 PM OMF File _ all data 64 channels_spl_001 01 omf 12 2 2013 9 34 PM OMF File _ all data 64 channels_spl_001 02 omf 12 8 2013 9 49 PM OMF File Desktop _ all data 64 channels_spl_001 missing9 omf 12 8 2013 10 25 PM OMF File Version 4 0 227 5 Menu Options Specify the details of the Multiple Files session After either a list of PL2 files or a single OMF file has been selected the Load Multiple Files dialog appears
263. l use cases The main Waveforms view uses newer graphics technology to render spike waveforms much faster than the Waveforms Legacy view However some older computers with older graphics hardware might not support the new graphics technology that the main Waveforms view now relies upon making the main Waveforms view unusable on those computers Using the Waveforms Legacy view is only recommended if the main Waveforms view fails to work properly 3 2 Info Grid Views This section describes the options in the Info Grid Views which consist of a family of similar views that display information in a spreadsheet format All of the Info Grid Views are grouped into an Info Grid Views sub menu under the View menu Version 4 0 25 3 Views and Displays The Sources view displays information about the Sources present in the currently loaded data file Sources are familiar to users of Plexon Omniplex System software They are ranges of channels that have shared characteristics such as sampling rate When a PLX or PL2 file created by the Omniplex System is loaded into Offline Sorter Software the Sources displayed should be those defined when the file was collected See Section 3 2 1 Sources View on page 27 The Channels view contains counts of waveforms for each unit in each channel The count of continuous samples is also shown See Section 3 2 2 Channels View on page 29 The Channel Parameters view contains channe
264. le exporting to 233 Numerics 2D and 3D Clusters views 23 52 54 128 2D Clusters view 61 32 bit and 64 bit versions 2 3D Clusters view 65 A About Offline Sorter 274 adaptive template algorithm 302 Adaptive Templates History view 37 algorithms spike sorting 10 aligned extraction 310 animate 238 animate eyepoint rotation 66 automatic waveform detection 249 B batch commands 235 313 324 binary file 110 224 C Channel Map view 37 channel mapping 286 loading 290 saving 291 Version 4 0 Channel Parameters view 30 281 Channels view 29 278 channels raw 277 clustering Bands method 168 Boxes method 163 K Means method 171 Lines method 165 methods 143 scanning method 182 semi automatic 157 Standard E M method 175 T Distribution E M method 178 Templates method 157 Valley Seeking method 176 Waveform Crossing method 153 Clusters view 2D 61 Clusters view 3D 65 Clusters views 2D and 3D 23 52 54 128 Clusters vs Time view 71 compatibility with other systems 8 continuous frequency spectrum 80 Control Grid 38 corresponding channels 282 Cross Correlograms view 91 customizing the Sorting Summary 87 D data channels 125 data files opening and importing 110 data flow 9 data converting data type 288 Index 1 data exporting 206 Davies Bouldin validity metric 309 definitions 16 Demo mode 5 digital filters 118 disable electrodes 294 distribution E M algorithm 305 Dunn validity metric 309 E Edit
265. le using the ForEachFile Scan and ForEachFile SaveScanResults batch commands This feature in combination with the automatic loading of SCAN files discussed above enables a convenient way of working with Offline Sorter Software which might be termed Supervised Automatic Sorting To use Supervised Automatic Sorting Version 4 0 215 4 Spike Sorting Step by Step you can set up a batch file to perform extensive sorting parameter scans on several large files and save the scan results to SCAN files This batch file can be run overnight as it could take hours to complete Then upon arriving at work in the morning you can simply open those files in Offline Sorter Software and the companion SCAN files will be automatically loaded for each channel You can then check each channel and use the Scan Graph view to select and inspect quickly each sorting within the scan You can quickly pick the best sorting and move on to the next channel i e by pressing the N key When the file is saved the picks for the best sorting for each channel will be saved This Supervised Automatic Sorting has several advantages it combines the thoroughness of scanning through automatic sorting with the safety of having a human in the loop to check sort quality but the checking can be done very quickly without having to sit and wait for each scan to complete 216 Offline Sorter Offline Spike Sorting Software Plexon Inc Chapter 5 Me
266. lgorithm Aligned Extraction 310 Enhanced PCA Feature Space 312 Batch File Processing 313 Batch File Commands 313 File 314 Dir 314 ForEachFile 314 ForEachChannel 315 Version 4 0 Set 317 Process 321 Example Batch Command File 321 Notes 322 Sample Batch Command Files 324 SortAndSave ofb 324 RemArtifactsSortAndExport ofb 325 ScanKMeans ofb 325 ScanValleySeeking ofo 325 FiltDetect ofo 325 SaveToNex ofo 325 Large File Tips 325 Prefer PL2 files 9325 Statistics Calculations 326 Buffer Sizes 326 Ignoring Continuous Data Channels 326 Initial PCA Calculation 327 Appendix A Navigating the Plexon User Interface A 1 Plexon User Interface A 2 Screen Elements A 3 Menus A 3 Toolbars A 5 Windows 6 Standard Menu Items and Dialogs A 11 Window Menu A 11 Run Menu A 13 Help Menu A 13 Messages Window A 16 Right click Menus A 17 Current Selections A 17 Undo A 17 Customization A 18 Toolbars Customization A 18 Commands Customization A 19 Keyboard Customization A 19 Run Menu Customization A 21 Options Customization A 22 Index xvi Offline Sorter Offline Spike Sorting Software Plexon Inc Chapter 1 Getting Started Ted metalan a crannies 1 2 Using the Sentinel USB Licensing Key cesceeeeeeeenees 1 LASS SING FUNCIONE 2s cc ctccess ssessssnobiaeessdioneeteatueecanceumetsediondes e HCl ic sa N navies aaRe 1 5 Off line Sorter Technical Support and Updates ceee 1 5 1 Offline Sorter T
267. line Sorter Software s primary purpose of sorting spikes but it can provide useful information such as whether the continuous data is contaminated by 60 Hz noise To display the Continuous Frequency Spectrum View Select the View Continuous Frequency Spectrum menu command or press the a button on the toolbar Conti nuous Frequency Spectrum The display power spectrum is the result of running an FFT on the samples that are currently displayed in the Timeline view starting at the left hand earliest time side The FFT is run on a maximum of 2048 samples so if the time range is such that many more samples are showing the power spectrum will be calculated from the first 2048 points as is the case in the above screenshot The vertical scale is always set such that the tallest peak fills the display vertically By default the frequency range displayed is 0 to one half of the sampling frequency for the continuous data Using right click menu items or the corresponding tollbar buttons the frequency range displayed can be expanded or shrunk moved higher or lower or reset to the initial range 3 13 Feature vs Feature View The Feature vs Feature view can be quickly used to view the feature space point distributions for many different features This can give a quick impression of which features might be most useful for sorting Offline Sorter Software divides the screen into boxes and each box contains a 2D Clusters view Much of
268. line are sorted into that unit 166 Offline Sorter Offline Spike Sorting Software Spike Sorting o amp x 0 Add Unit Delete Unit Add Wfs Remove Wfs Invalidate Wfs E Note that this generally results in too many waveforms being included into the new unit So the unit definition should be refined by adding additional lines that the waveforms are required to intersect 3 Add additional lines Pressing the first button on the secondary toolbar circled red in the above screenshot will again change the cursor to a drawing hand and allow another line to be defined Note that after drawing the line the waveforms are re sorted so that waveforms sorted into the unit pass through all defined lines for the unit Version 4 0 167 4 Spike Sorting Step by Step o amp x Fos i Add Unit Delete Unit Add Wfs Remove Wfs Invalidate Wfs Repeat this step as desired to add additional lines to the unit definition 4 Adjust the lines Each small box on the screen can be grabbed and dragged with the mouse to reposition the lines Individual lines can be removed by selecting them and pressing the second button on the small toolbar The third button on the small toolbar controls whether when multiple Lines units are defined the lines for all units or just for the currently selected unit show up on the display Note The Lines sorting algorithm is greedy in that a waveform is sorted into the first unit
269. lowed by a colon followed by the shift of the second unit template The total number of Overlap Templates is shown on the left under Overlap Templates Because slight overlaps between waveforms where the overlapping waveform occurs late in the first waveform are difficult to resolve the Overlaps Analysis Version 4 0 101 3 Views and Displays algorithm allows the specification of a Minimum Overlap value which is specified in the number of samples When constructing the Overlap Templates the starting point for the overlaps as in the least amount of overlap between the two unit templates is this Minimum Overlap In the above example the waveform length is 32 samples but because the Minimum Overlap is 10 samples the most shifted Overlap Template has the second waveform starting at 22 samples Note also that the waveforms get closer to complete overlap the overlap tends to interfere with the threshold crossing position which means that effectively both unit templates could be shifted relative to their non overlapping position The blue text can be hidden by unchecking the Show Unit Shift Text checkbox The Show on Clusters Views checkbox can be used to examine the effects of overlapping waveforms in feature space Since each of the Overlap Templates is just a waveform it corresponds to a point in feature space just like any other waveform That is Offline Sorter Software can calculate the feature space values for each of the
270. ls and Sources Another way to select another channel to view is to use the Showing button in the Timeline view Spike Ch 3 SPKOS Control Displays C Center Selected Showing which brings up a dialog where the user can select any channel from any other continuous Source to show j Timeline Channels to Display Showing Source Channel Spike SPK Chs 1 64 x 3 SPKO3 z Continuous FP Chs 193 256 v 195 FP03 Z lt I lt Version 4 0 285 6 Reference Topics 6 2 Channel Mapping Offline Sorter Software has the concept of a channel which has a name and a number An Offline Sorter Channel can potentially contain both spikes and continuous data Neural data files also have a concept of a channel which generally also has a name and a number We will refer to the channels in the neural data file as Raw channels A Raw channel is always either a Spike channel or a Continuous channel but not both The purpose of the Info Channel Map tab is to display and allow control over how the Raw channels from the neural data file are assigned to Offline Sorter Channels Note Channel re mapping is supported only for Plexon PLX files An Offline Sorter Channel displays data from a raw channel from a spike Source or from a continuous Source or from both Each Source is considered to have a Data Type which is
271. ly the filter and close the dialog box Note From the Timeline view it is only possible to apply a low cut filter to the continuous data But from the Filter Continuous Data dialog it is possible to apply a high cut filter instead of a low cut filter by selecting the High cut radio button This can be used to obtain LFPs from a wideband continuous signal by removing the high frequency spike activity The same filter parameters apply to the high cut filter except the sense of the filter is reversed to remove the high frequency signal energy instead of the low frequency signal energy Version 4 0 119 4 Spike Sorting Step by Step 4 2 6 Specifying the Waveform Detection Options The Total Waveform length and Prethreshold Period parameters specify the time range of the waveform segments extracted from the continuous record The Dead Time is the minimum time between extracted waveform segments For example if the DeadTime 1 msec and the recorded signal crosses the threshold at times t 5000 0 msec and t 5000 8 msec then only one waveform segment aligned around the t 5000 0 msec crossing is detected and extracted If the DeadTime 0 5 msec however then two waveform segments are extracted one aligned around the t 5000 0 msec crossing and the other aligned around the t 5000 8 msec crossing Note that these waveforms can actually overlap depending on the Total Waveform Length Some of the same samples might be included int
272. lysis View on page 99 and Section 4 6 5 Rescuing Overlapping Waveforms on page 194 L Ratio and Isolation Distance sort quality metrics were added See Section 4 7 2 2 Statistics Feedback While Sorting on page 203 Detailed information about a number of menu options was added See Chapter 5 Menu Options Information was added about opening multiple PL2 files See Section 5 1 3 File Open Multiple Files on page 226 Information was added about exporting PL2 files See Section 5 1 8 File Export to New PL2 on page 232 The Digital Referencing option was added in the Waveforms menu See Section 5 5 11 Waveforms Perform Digital Referencing on Continuous Data on page 246 Reference information was added regarding channels and sources viewing a spike channel together with a continuous channel channel mapping stereotrode and tetrode data Median Absolute Deviation MAD technique sort quality metrics the thresholding algorithm enhanced principal components analysis PCA feature space and other features See Chapter 6 Reference Topics Offline Sorter Offline Spike Sorting Software April 2009 Version 3 0 of the Offline Sorter User Guide issued February 2006 Version 2 8 of the Offline Sorter User Guide issued January 2005 Version 2 7 of the Offline Sorter User Guide issued September 2004 Version 2 6 of the Offline Sorter User Guide issued March 2004 Version 2 5 of the Of
273. mand selected in the Commands area Description This area displays a description of the currently selected command in the Commands area e Assign This button assigns the shortcut in the Press New Shortcut Key area to the selected command in the Commands area If the shortcut key is already assigned to another command a confirmation dialog box displays to allow or cancel the reassignment e Remove This button removes the selected shortcut key in the Key Assignments area from the selected command in the Commands area e Reset All This button removes all custom key assignments A confirmation dialog box displays to allow or cancel the operation A 20 Offline Sorter Offline Spike Sorting Software Procedure for Customizing Keystroke Shortcuts 1 From the Window menu select Customize and then click the Keyboard tab of the Customize dialog box 2 Choose a category from the Category dropdown and from the Commands list select the command that you want to bind to a keystroke shortcut 3 If there is already a key assignment listed in the Key assignments area you may remove it by clicking the Remove button if you wish You may also just reassign a new key combination to the selected command See Step 5 4 Click the mouse in the Press new shortcut key area Click the CTRL or ALT key and hold it down while clicking another key The dual key combination will be displayed in the Press new shortcut key area This com
274. matching waveforms by creating two waveforms for each overlap The rescued Version 4 0 197 4 Spike Sorting Step by Step waveforms will be sorted into the correct units as determined by which unit permutation produced the Overlap Template that matched the waveform and the waveforms will have timestamps that are determined by the Unit Shifts corresponding to the Overlap Template 4 7 Examining the Sorting Offline Sorter Software offers many ways to inspect and examine the sorting done to a channel to be sure that it is of sufficient quality 4 7 1 Waveform Inspection After units have been sorted individual waveforms can be compared with the template average waveform of each of the sorted units in the Waveform Inspection window The waveform inspection process involves three steps 1 Activating the Waveform Inspection Window page 198 2 Selecting a waveform either by Selecting Waveforms in the Timeline View page 199 or by Selecting Waveforms in the Clusters Views page 199 3 Comparing the Waveform with the Templates page 199 Activating the Waveform Inspection Window Select the View Waveform Inspection Window menu command or za press the S button on the toolbar Info BE Inspection 198 Offline Sorter Offline Spike Sorting Software Examining the Sorting 4 7 1 1 Selecting Waveforms in the Timeline View Left clicking in the Timeline view near a waveform will select that waveform d
275. mmonly used functions in various views You may print it by clicking Print or close it by clicking OK Version 4 0 A 13 Appendix A Navigating the Plexon UI Quick Reference Marker Types T Time Only just a tine F Frame tines of video franes I Interval Start and End times S Scalar time and a single value E Coordinates time and X and Y data values L Ievel Interval times of Scalar level crossings z Zone Interval times vith Coordinates in zones Mouse and Keyboard Shortcuts All Views Space Return Toggle Play Pause Left Right Arrow Previous Next Frame Up Down Arrow Increase Decrease Animation Speed Esc Cancel Marker add node Activity View left Click in left Drag divider or Drag scroll up and down Left Click in top Drag divider or Drag current frame marker Left Click in view Select Marker Occurrence Shif t Left Click Drag Selected Marker Occurrence Left Double Click Select Marker Right Click Context Menu Mouse wheel Scroll up and down Markers View Left Click Select Marker or change Show flag Display Color Right Click Context Menu Mouse wheel Scroll up and down Channels View Lef t Click Change Show Lef t Double Click Select Channel Right Click Context Menu Mouse wheel Scroll up and down Marker Data View Lef t Click Select Occurrence Right Click Context Menu Mouse wheel Scroll up and down Video View Left Click depends on mode Right Click Context Menu e
276. mplification gains 30 Offline Sorter Offline Spike Sorting Software Info Grid Views By right clicking in the Channel Parameter view and selecting Adjust Gains the gain values can be changed for a channel See Section 4 2 4 Adjusting Gains on page 115 Filter is for PLX and PL2 files only and it indicates if the filter is switched on for the channel Threshold shows the threshold value in A D counts and the associated voltage that was used to collect the waveforms For continuously recorded data files the threshold for the currently selected channel can be changed by typing a new value Zoom refers to the scaling factor that Offline Sorter Software uses to display the waveforms in the Waveforms view and cluster display The scaling factor for the currently selected channel can be changed by typing a new value SNR Signal to Noise Ratio is only displayed for a channel if spike extraction from continuous data has been performed on that channel See Section 3 21 Threshold Scan Graph on page 97 LC Filter the type of low cut filter that has been applied to continuous data in the channel Poles the number of Poles in the low cut filter shown regardless of whether a filter was actually applied Cutoff the cutoff frequency of the low cut filter shown regardless of whether a filter was actually applied Note The currently selected channel is designated with an gt gt in the
277. ms and also set some important parameters for how each sorting algorithms behaves same as changing these parameters from the Control Grid or Tools Options Sort The tool also supports the selection of whether to perform the sorting in 2D or 3D feature space and whether to apply the automatic sorting only to the currently loaded channel or to all channels in a selected Source If units already exist the K Means Standard E M and T Distribution E M support running where the existing units can be used as the starting point for sorting This is called Continue sorting for lack of a better name Checking the Perform Continue Sorting checkbox will sort using the existing units as a 252 Offline Sorter Offline Spike Sorting Software Sort Menu starting point Note that this checkbox is only available when units exist and one of the supported sorting methods is selected Pressing the Apply button will perform the selected sorting to the current channel only even if the All Channels in Spike Source radio button is enabled and will keep the dialog open which is useful for exploring different sorting methods and parameters An Undo Previous Sort button will also revert the current channel only back to its state before the sorting was applied Pressing the OK button will then perform the automatic sorting as currently configured in the dialog potentially to all channels in the selected Source 5 6 3 Sort Perform Tem
278. n 6 4 54 Offline Sorter Offline Spike Sorting Software 2D and 3D Clusters Views Features Available for Sorting on page 294 To control which features are active use the Active Features tab in the Tools Options dialog box General Feature Calc Sot Templates Stats Refractory Period Colors Display Printing Options Active Features Cache Offline Sorter can calculate more waveform features than can be conveniently displayed in the feature selection droplists This dialog allows control of the set of active features Features with EL in them refer to individual Electrodes for Stereotrode Tetrode data These features will not be displayed in the feature droplists unless Stereotrode Tetrode data is being viewed Active Features Available Currently Active PC 4 a a PC5 PC2 PC6 E PC3 PC7 La Slice 1 PC8 Slice 2 Peak FWHM Peak Valley FWHM Adi zam y Peak Tick Peak Valley Valey Tick a Nonlinear Energy Slice 3 lt lt Remove Timest SLs Peak to Valley Ticks PC2EL1 Valley to Peak Ticks ____ Are Sqrt Energy Peak EL 1 ISI Previous Valley EL 1 ISI Next PC 1EL2 Average PC 2EL2 Peak Valley EL 1 Boo ci 2 Peak Valley EL 2 peace Hove Up Move Domn The currently active features appear in the right hand list box and the currently inactive and thus available for activation features appear in the
279. n Overlaps Analysis button a Some overlap templates did not cross threshold message may appear This means that one 196 Offline Sorter Offline Spike Sorting Software Unit Cleaning or more superimposed overlap templates did not reach the threshold These overlap templates that failed to cross threshold are ignored for the rest of the analysis and are shown as flat lines with unit shift values of 1 1 in the Overlaps Analysis grid Inspect and Clean the Overlaps Some of the Overlap Templates are not convincing just because of how the unit templates happened to superimpose to form the Overlap Template The user should inspect the Overlaps that matched the Overlap Templates the waveforms shown in yellow to make sure they make sense as overlaps To facilitate this left clicking and holding in one of the Overlap Templates grid cells that has yellow overlap candidate waveforms will select and highlight that candidate overlapping waveform and show it in the Waveforms and Clusters views You should inspect the Overlap Templates grid for all unit permutations using the Next gt and lt Prev buttons The candidate overlapping waveforms that are not acceptable and should not be rescued can be removed from consideration by right clicking in the grid cell for the overlap and selecting Remove Matches for this Overlap Template from the context menu If all matches for a particular unit permutation are not acceptable they ca
280. n all be removed by selecting Remove Matches for all Overlap Templates from the context menu Conversely it is useful to inspect whether a particular waveform shown in the Waveforms or Clusters views will be rescued or not Clicking on a waveform in the Waveforms or Clusters views will display that waveform in all cells in the Overlaps Analysis grid Also it will find the closest Overlap Template in any unit permutation to the waveform and highlight the cell for that Overlap Template in the Overlaps Analysis grid The cell will be highlighted with a green box if the selected waveform matched the template to within Tolerance and therefore will be rescued and the cell will be highlighted with a red box if the selected waveform did not match the Overlap Template to within Tolerance Note that the best Overlap Template is always found and shown but even the best matching Overlap Template might not have matched to within Tolerance In either case the Tolerance that would be required in order to match the selected waveform to the Overlap Template is shown in white text in the upper left corner of the cell This is useful to determine why a waveform that may look like an overlap in the Waveforms view did not match any of the Overlap Templates and or it can provide insight into what to set the Tolerance value to in order to detect and rescue what seems like overlaps Rescue the Overlaps Pressing the Rescue Overlaps button will rescue the overlap
281. n or hidden by selecting Show PCSTs from the right click menu and they can be made more or less transparent by pressing the I Fade In or O Fade Out keys 5 5 5 Waveforms Use Only Currently Visible Waveforms This is a toggle able menu item When it is selected checked the sorting and other waveform processing functions within Offline Sorter Software will act only on those waveforms that are showing in the Waveforms view That is any waveform that is not showing in the Waveforms view perhaps due to the View only selections discussed in previous sections will not participate in or be altered by the Offline Sorter functions Selecting this menu item is equivalent to pressing the E button in the toolbar Version 4 0 245 5 Menu Options 5 5 6 Waveforms Use Only Unsorted Waveforms This is a toggle able menu item When it is selected checked the sorting and other waveform processing functions within Offline Sorter Software will act only on the Unsorted waveforms That is any waveform that has been sorted into a Unit will not participate in or be altered by the operations Selecting this menu f 7A item is equivalent to pressing the ci button in the toolbar 5 5 7 Waveforms Invalidate Waveforms Selecting this menu item will put the Waveforms view and Clusters views into the mode where the user is expected to cross on the Waveforms view or circle on the Cluster views the waveforms that should be
282. n page 233 4 8 3 Exporting Information Offline Sorter Software has powerful and flexible facilities to export information for further analysis Offline Sorter Software can export information to the following destinations e Text files you can choose the delimiter between values e A MATLAB file The data appears as a matrix variable when opening the file in MATLAB This option is only appears MATLAB is installed on the same computer as Offline Sorter Software e A Microsoft Excel spreadsheet This option only appears if Microsoft Excel is installed on the same computer as Offline Sorter Software During the information export Excel opens and accepts the exported information into the first sheet on the spreadsheet You can then save the spreadsheet from within Excel Offline Sorter Software can export three varieties of information e Per Waveform information via the File Export Per Waveform Data menu selection This item generates a line of information a row for each waveform that satisfies the export criteria The kind of information items that can be exported per waveform include the timestamp unit assignment and the values of any feature e Per Channel information via the File Export Per Channel Data menu selection This item generates a line of information for each channel in the file The kind of information items that can be exported per channel includes the numbers of waveforms and units and the Sorting Quality St
283. n table shows the list of PL2 files that will be loaded along with the start time and date that the file was acquired and the duration of the file Note that the specified files are always sorted in chronological order Files can be added to or removed from the list using the buttons below the table To select files for removal click on the leftmost number column for each file so that the entire row is highlighted The first file in the list is loaded normally into Offline Sorter Software but all subsequent files are loaded and placed artificially on the timeline That is the timestamps in subsequent files are artificially offset so that they appear sequentially on the Timeline view When files are saved this artificial offset is removed from the timestamps before the data is saved There are three options for how to artificially place the subsequent files on the timeline e Create a time gap allows the user to specify a time gap 1 second by default between each gap This is a convenient way to clearly indicate where files begin and end e Create the time gaps according to the file start times this will place subsequent files on the timeline according to their actual file start times This option is only useful for files that were acquired with relatively short intervals between the files Using this option when the files are separated in time by more than a few minutes is not recommended e No time gaps between files
284. ndicated by the Type droplist to be performed at each step of the parameter over the range specified by the Start End and Step By values At each step the Sorting Quality Statistics are calculated and displayed in a table along with the number of units and percentage of waveforms sorted By default Offline Sorter sorts and calculates the Sorting Quality Statistics in 2D feature space but the 3D feature space can be used instead by checking the Use 3D checkbox When the scan completes the best values of the parameter being scanned according to each of the Sorting Quality Statistics is displayed in a table To re sort the channel with any of these best values click Re Sort Using This Value on the line for the statistic Also double click on any line in the scan table to re run the procedure using that value of the scanned parameter For some of the sort quality metrics smaller values are better while for others larger values are better And the magnitude of the numbers calculated varies Version 4 0 179 4 Spike Sorting Step by Step widely which makes it difficult to plot different metrics on the same graph see the table below To address these problems Offline Sorter Software can also calculate and display a second normalized version of each sort quality metric The normalized version of a sort quality metric is just a simple mathematical function on the original sort quality metric
285. ne Sorter Channel actually has the ability to hold either a spike channel a continuous channel or both For stereotrode tetrode data a single Offline Sorter Channel can hold 2 or 4 spike channels 2 or 4 continuous channels or 2 or 4 of each type The only time that a single Offline Sorter Channel will contain both spike and continuous data is after waveform detection has been performed on a continuous data channel see Section 4 2 6 1 3 Offline Sorter Channels and Sources Immediately after a data file is loaded every Offline Sorter Channel will be affiliated with just a single Source that Source will be a spike Source if the 278 Offline Sorter Offline Spike Sorting Software Details of Channels and Sources Offline Sorter Channel has spikes or a continuous Source if the Offline Sorter Channel has continuous data When an Offline Sorter Channel is selected as marked with a gt gt next to the channel in the Channels view Offline Sorter Software will mark the corresponding Source in the Sources view with a C gt for continuous or S gt for spike For example this screenshot shows the Sources view after loading a data file with 64 Spike channels SPK 64 continuous 40Khz wideband channels WB 64 continuous 40 Khz filtered channels SPKC and 64 continuous 1 Khz field potential channels FP with Offline Sorter Channel 129 selected as the current channel 2500 0000 m 8 1 64 ae He 25
286. ne Sorter Software as they will on Sort Client check the Use MAP compatible Fit Tolerance checkbox on the Tools Options Templates tab Let waveform w w wN template t t1 tN where wj and t values are in AD units typically between 2047 and 2048 N digitized data points 300 Offline Sorter Offline Spike Sorting Software Details of the Sorting Algorithms Note For Plexon MAP users The MAP system has 12 bit A D conversion 24 12 4096 Therefore the AD values of waveform and template amplitudes are integers between 2047 and 2048 The difference measure dw between a waveform and a template is the square root of the mean sum of square error 1 dy Jp t 2 where M is the sort width for the current channel in terms of the number of data points represents a sum over i the samples in the sort width For stereotrode and tetrode data the sum is I S 2zi t where the first summation is over the electrodes The waveform is assigned to a template s unit if Template Fit Tolerance lt dy The waveform is assigned to the first starting with Unit a template that satisfies the above relation 6 5 3 MAD Calculation When calculating band tolerances or unit Fit Tolerances Offline Sorter Software can use a modified calculation of the standard deviation that purports to be less sensitive to outliers This modified standard deviation relies on the MAD median absolute devia
287. nel re mapping has been performed a neural data file loads with a default channel mapping scheme The Channel Map tab only displays the Offline Sorter Channels for a single Source at a time Likewise if channels are to be re mapped each Source must be mapped independently The For Source droplist highlighted in screenshot below can be used to select which Source to display and map Version 4 0 287 6 Reference Topics 6 2 1 Stereotrode and Tetrode Channel Mapping The following screenshot shows a standard mapping of 16 Raw spike channels into four tetrode Offline Sorter Channels For example the tetrode channel named SPK09 consists of Raw spike channel 9 as electrode 1 Raw spike channel 10 as electrode 2 etc Note The Neuralynx file format stores stereotrode tetrode data such that a single Raw channel contains the data for all electrodes in the stereotrode tetrode data In this case the Channel Map tab can be misleading 6 2 2 Converting Data Type An advanced feature in Offline Sorter Software lets you force interpret spike data to be any data type For example data files that were acquired as single channel 288 Offline Sorter Offline Spike Sorting Software Channel Mapping data can be treated as if they were stereotrode or tetrode data To convert the Data Type use the droplist in the second row For Source SPK Chs 1 16 Single E Stereotrode As soon as the Da
288. neric Neuroshare Import section Then load any arbitrary data file using the selected Neuroshare DLL and the File Import File via Neuroshare menu item Note For any files imported using a Neuroshare DLL the Neuroshare Segment entities become spike channels and the Analog entities become continuous channels 5 1 3 File Open Multiple Files Offline Sorter Software can open multiple PL2 files that were acquired over a period of hours or weeks and essentially treat them as a single file as far as sorting and analysis are concerned This is useful to ensure that a set of files are sorted and analyzed consistently and is much more convenient that applying the sorting done in one file to another file via TPL files The PL2 files that are loaded together must be compatible with each other in that they have the same Sources and Channels structure The underlying assumption is that all the PL2 files that are to be analyzed together were acquired using the same experimental setup but at different times After sorting the Save functionality is available it will Save a new version of each individual loaded PL2 file Offline Sorter Software contains an OMF file format that remembers the list of PL2 files that should be loaded together along with some additional information After an OMF file is saved it can be selected and loaded as if it were a supported file format in Offline Sorter Software loading the OMF will result in al
289. ng algorithm over the range of D O F Multipliers specified The D O F Multiplier affects the sorting as described in Section 6 5 Details of the Sorting Algorithms on page 299 Scan number of clusters for K Means This scan type runs the Semi Automatic Clustering using K Means page 171 sorting algorithm scanning the number of clusters over the range specified by the Start End and Step By values The initial cluster centers are automatically placed 180 Offline Sorter Offline Spike Sorting Software Spike Sorting according to the Tools Options Sort Seed clusters pattern radio button the cluster centers can be either e Arranged evenly along a diagonal line passing through the feature space mean point the average of all the waveforms or e Arranged evenly in a circle around the feature space mean point The K Means clustering is known to be sensitive to the initial cluster centers so manual placement of the K Means cluster centers may lead to better results than those produced by the K Means scan The Scan Random Seed for K Means scanning method described below can be used to explore the degree of sensitivity of the K Means sorting results to the initial placement of cluster centers Note The current setting of the Outlier Threshold in the Control Grid is used in the K Means sorting at each step Scan Number of Clusters for Std E M This scan type runs the Semi Automatic Clustering using Standard E M pa
290. ng this one are repeated for each Source in the file e The second line which is the first line of the per Source block is the Source ID This must match a Source ID that is currently loaded into Offline Sorter Software e The next line contains two numbers separated by commas The first number N is the number of Offline Sorter Channels described in the mapping The second number is the trodalness T of the mapping 1 for single electrode 2 for stereotrode or 4 for tetrode The example that follows represents four channels mapped as tetrodes N T 4 4 e The next N lines describe the raw channels mapped to each Offline Sorter Channel Each line consists of the Offline Sorter Channel number which must be greater than or equal to 1 followed by a tab or space s then followed by a comma separated list of T raw channel numbers The position within the comma separated list determines the specific electrode within a 290 Offline Sorter Offline Spike Sorting Software Stereotrode and Tetrode Data stereotrode or tetrode In the following example channel 4 has electrodes 13 14 15 and 16 as a tetrode Sample channel mapping file contents Number of Sources 1 Source ID 7 4 4 1 1 2 3 4 2 5 6 7 8 3 9 10 11 12 4 13 14 15 16 6 2 4 Saving a Channel Map to a File Although ofm files can be manually created from scratch using a text editor a better way is to have Offline Sorter Software export an ofm file that is
291. nits This can provide a less cluttered view of the waveform data Vavetorms Be POE A ELE AA bean a ee ey ee ee ee ee ee es 46 Offline Sorter Offline Spike Sorting Software Waveforms View If the number of waveforms in a channel exceeds the Max w f draw value in the Control Grid the Waveforms view and the Clusters displays draw only every Nth waveform where the divisor N is calculated to bring the number of drawn waveforms under the Max w f draw limit This ensures that drawing occurs with an acceptable speed on slower computer systems When this decimation is in effect Offline Sorter Software highlights the wf count text in red In the preceding screen shot the Max w f draw is set to 1000 The divisor N in this case is 6 as shown in the red highlighted section which indicates that only every 6th waveform is being drawn When the Control Displays checkbox in the Timeline view is checked decimation is disabled and all waveforms in the selected time interval are displayed CAUTION Sorting During Decimation If sorting is performed while decimation is occurring it can result in lost information If possible increase the Max w f draw setting so that decimation does not occur The Use only currently visible waveforms setting in the Tools Options General menu dialog controls whether waveforms that are not being drawn are included in Waveform Crossing and Contour sorting Version 4 0 47 3 Vie
292. not all the waveforms for the channel To select which waveforms are shown in the Waveforms view use the Waveform View parameter in the Control Grid To remove some of the waveforms from a particular unit 1 In the Control Grid change Waveform View parameter value to Sel Unit s Selected Units 2 To select the unit click at the unit s template display amp Add Unit Delete Unit Add Wf Remove Wfs Ainvalidate Wf 4 Move the mouse pointer to the Waveforms view wy The pointer changes to a drawing hand 188 Offline Sorter Offline Spike Sorting Software Unit Cleaning Click the left mouse button and drag a straight line across the waveforms to be excluded from the unit After several iterations of clicking Remove Wfs and drawing a line the unit can appear as follows Adding or Removing Waveforms using other views Note Waveforms can be added or removed from the selected unit by circling them in the Clusters views after pressing the Add Wfs or Remove Wfs buttons or e by selecting the Units Add Waveforms to the Selected Unit or the Units Remove Waveforms from the Selected Unit After selecting one of these commands move the pointer to the Clusters view Note The pointer changes to a blue drawing hand ro for adding waveforms and a red drawing hand 3 for removing waveforms Click the left mouse button and drag a line around the units
293. not save the inverted waveforms 4 4 1 2 Clusters Views Section 3 7 2D and 3D Clusters Views on page 52 shows the waveforms as points in feature space You can choose to use either the 2D Clusters view or the 3D Clusters view or both The variables shown on the X Y and Z axes can be chosen from a list of active features For more information see the Axis Feature Selection section under Section 3 7 2 Clusters Views Common Functionality on page 54 By default the Clusters views show the PC1 PC2 and PC3 features on the X Y and Z axes References 2D Clusters View 128 Offline Sorter Offline Spike Sorting Software Preparation For Sorting References 3D Clusters View 4 4 1 3 Timeline View The Timeline view shows the waveforms in their temporal sequence This is useful for determining if the data comes in bursts or if there are gaps in the data Also the times of occurrences of external events can be superimposed on the Timeline view See Section 4 4 3 Examining Events on page 137 which allows the spike activity surrounding those events to be seen _ Spike Ch 132 SPKCO4 Sh I ied Cont Ch 132 SPKCO4 ta E Control Displays F Center Selected li jh il i i 1 l i I W Zoom 1 0 2 50 mV Th 7 94 498 pv Detect Waveforms Low cut Filter 4 pole Cutoff Freq 250 Hz Apply Scroll Range 10 0000 0 0000 4 4 2 Invalidating Waveforms Offline So
294. not shifted at all You can perform the same alignment operation on all channels in a Source instead of just on the current channel To do so select Waveforms Align When the same Align Waveforms dialog box as shown in Step 2 on page 4 140 appears click All Channels in Spike Source and select the desired Source from the droplist Now the alignment operation is applied to all of the spike channels in the Source 4 4 5 Adjusting the Sort Range Often the extreme tail and the earliest samples of the waveforms are not useful in separating units from each other or separating the units from noise To specify the range of samples within the waveform that is used to calculate the Principal Components See Section 6 4 1 Principal Components on page 297 and used in feature calculation and Template Sorting adjust the Sort Start and Sort End parameters in the Control Grid see the following illustration to locate the parameters When they are visible you can adjust the Sort Start and Sort End times by dragging the corresponding vertical line with the mouse see the double ended arrow in the following illustration Ch_01 2500 my Contours ClustHist Max 10 Outlier Thr 11 30 Sort End View wif All Cluster View All 1000 usec The Sort Start and Sort End time point is the lower and upper bound respectively of the time interval of the waveform used to calculate principal components feature values and for Template Sorting
295. ns on the raw channels within a stereotrode tetrode channel If the Compensate for gain differences with Stereotrode and Tetrode channels checkbox in the Tools Data Import Options Plexon dialog is checked Offline Sorter Software will alter the waveforms in the subsequent raw channels to what they would be if that channel had the same gain as the first raw channel Note that this is a digital adjustment of the actual waveform values and it can either increase or decrease the waveform values depending on the ratio between the first raw channel gain and the gains of the subsequent raw channels 6 3 2 Extracting Stereotrode or Tetrode Spikes from Continuous Data As outlined in Section 3 11 4 Stereotrode and Tetrode Waveform Detection on page 78 Offline Sorter Software can extract spikes from continuous data channels that are either loaded as stereotrode tetrode channels or mapped to stereotrode tetrode channels using the Channel Map tab When a stereotrode 292 Offline Sorter Offline Spike Sorting Software Stereotrode and Tetrode Data tetrode channel has been loaded the Continuous Data Display shows the two or four continuous data traces stacked vertically on top of each other If a low cut filter is applied it applies to all traces in the stereotrode or tetrode channel For waveform detection on stereotrode tetrode continuous data a threshold crossing on any electrode results in the cap
296. nsideration It can be visualized as the radius of an ellipsoid in feature space that contains an equal number of within unit spikes and non unit noise spikes Isolation Distance is therefore not defined for situations where a unit contains more spikes within it than exist outside of the unit Large values of Isolation Distance indicate a well separated unit the ellipsoid radius had to be made very large to include as many non unit spikes as there were within unit spikes See the reference listed below for the mathematical definitions of these quantities The per unit values of L Ratio and Isolation Distance appear in the Per Unit Statistics section of the Stats option in Info Grid Views in Offline Sorter L Ratio is sometimes abbreviated as just LRatio within Offline Sorter Software and Isolation Distance is sometimes abbreviated as IsoDist within Offline Sorter Software Since machinery already exists within Offline Sorter Software to select and display per channel sort quality metrics Offline Sorter Software has added two new per channel sort quality metrics based on these per unit sort quality metrics e Worst LRatio the largest and therefore worst L Ratio value for any unit in the channel e Worst Isolation Distance the smallest and therefore worst Isolation Distance value for any unit in the channel These then show up in Offline Sorter Software where the other per channel sort metrics listed in the previous sect
297. nt After selecting this the cursor changes to horizontal arrows and the new time segment can be defined by click dragging on the timeline view The newly defined time segment is selected as the current time segment Add New Time Segment Same as the above except that the new time segment is added on to the end of the list of existing time segments Invalidate Waveforms in Current Segment A convenience function to invalidate all waveforms within the currently selected time segment This is intended for use after using the above menu items to define a new time segment This provides a convenient mechanism to invalidate all waveforms within a noise episode where the user has just created a new time segment that demarcates the noise episode Mark Invalidated Waveforms in Current Segment as Valid A convenience function that is intended mainly as an undo mechanism for the above function Show Time Segment Boundaries if checked show vertical lines and labels at the boundaries between time segments Zoom Time Range to Selected Segment Set the displayed time range to show the entire current time segment See Section 3 2 5 Time Segments View on page 34 Undo Last Zoom reverts to the time range previously in effect before the last zoom of any kind was performed in the Timeline view Show Zero Line for Continuous Data if checked shows a dashed line representing 0 volts Version 4 0 51 3 Views and Displays e S
298. nter of gravity of all unassigned points are also included in the cycle For example if a channel contains two 2 sorted units pressing the c or L keys moves the Look At point as follows CG of All points gt CG of Unit a points gt CG of Unit b points gt CG of all unassigned points Resetting the Eyepoint and Look At points Pressing the left and right mouse buttons simultaneously resets the eyepoint and Look At points to their defaults looking directly down the Z axis with the Look At point at the center of gravity of all points Showing Ellipsoids The 3D Clusters view can display ellipsoids around clusters by selecting the Show Ellipsoids right click menu command or by pressing the E key while the 3D Clusters view has input focus The ellipsoids are oriented along the principal axes of the cloud of points with the lengths of the ellipsoid axes being the standard deviation sigma of the points along each axis scaled by the value of the Outlier Threshold in the Control Panel For example the ellipses shown below show the 3 sigma boundaries around each unit because the Outlier Threshold was set to 3 Offline Sorter Software draws the ellipses Version 4 0 67 3 Views and Displays semi transparently in the color associated with the unit Observe that the points both inside and outside the ellipsoid can be seen Run Valley Seeking f SA Delete Selected Unit BJ 2D Clusters BE 3D Clusters The transparency of
299. ntrol Grid The voltage from zero to peak appears to the right of the Zoom control Thr This is the voltage threshold control for waveform extraction the threshold voltage level is shown to the right of the Threshold control and it is represented by a solid horizontal line in the window display The threshold can be adjusted by dragging the horizontal line up or down with the mouse or by moving the slider to the left of the stripchart This item is enabled only if the data is continuous Detect Waveforms Options Click this button to display a dialog box for defining the waveform detection option settings such as the Total Length Prethreshold Period Dead Time for the waveforms extracted Signal to be thresholded Raw Energy Nonlinear Energy and the Energy Window Width See Section 4 2 6 Specifying the Waveform Detection Options on page 120 This item is enabled only if the data is continuous Detect Waveforms Apply Click this button to extract waveform segments around threshold crossings using the current waveform detection option settings This item is enabled only if the data is continuous Low cut Filter These droplists select the type Butterworth Bessel or Elliptic and the number of Poles for the low cut filter applied to the continuous data Changing these items will enable the Apply button discussed below These items are enabled only if the data is continuous Cutoff Freq and Apply The Cutoff F
300. nts PC3 PC8 The detailed shape of the PCA eigenvectors are not always straightforward to interpret Generally the first principal component the yellow line is largest in those sections of the waveform where the most variance is The shapes of the higher principal component eigenvectors are constrained by the fact that all the eigenvectors must be orthogonal to each other which makes their interpretation difficult Note though that they approach zero at the trigger point e g at a time value equal to the prethreshold into the waveform All waveforms have an amplitude equal to the trigger threshold value at this point in the waveform with essentially no variance Thus all the eigenvectors are zero at that point 3 16 ISI Histograms View The ISI Histograms view provides a larger printable version of the Inter spike Interval Histograms that appear in the Cluster Display The range for the ISI Histograms is 0 zero to the ISTHistMax ms Control Panel setting 90 Offline Sorter Offline Spike Sorting Software Cross Correlograms View To display the ISI Histograms view Select the View ISI Histograms menu command or press the button on the toolbar There are several options for displaying the ISI Histograms from the right click context menu The view can be configured to show the ISI Histograms from all sorted units from all sorted units plus an ISI Histogram for the unsorted waveforms or only for the currently
301. nts can be created via the Create New Time Segments dialog see Section 5 5 4 Waveforms Create Time Segments on page 243 during Interval Selection see Section 5 8 6 Tools Interval Selection on page 264 or from the Timeline view see Section 3 6 2 Other Features of the Timeline View on page 51 Time segments may also be imported from NeuroExplorer file Intervals and exported to NEX files see Section 5 1 9 File Export to NEX on page 233 34 Offline Sorter Offline Spike Sorting Software Info Grid Views 0 000000 _29 481725 0 000000 2948173 l E i 113 053952 482 99622 0 0822919 5 28 2 948173 5 896345 i i i 122 346496 469 27643 0 0974541 4 11 5 896345 8 844518 i i 16 118184 1224 9820 0 0899648 4 951 8 844518 11 792690 H i 30 041203 901 23609 0 0791368 6 28 11 792690 14 740863 H 122 324049 714 36957 0 0889626 4 79 14 740863 17 689035 E 110 640111 266 00278 0 1314480 3 27 17 689035 20637208 E 120 147644 402 95289 0 08731 16 4 98 23 585380 26 533552 E i 19 3687260 365 38031 0 1081490 3 641 9 Segment 10 26 533552 29 481725 q i i 19 381168 988 43961 0 0954366 4 75 The Segment column shows the editable name of the time segment The Start and End columns show the extent of the time segment in seconds The Show column controls whether the segment boundaries are shown on the Timeline view and the Display Color control
302. nu Options AM gil AL Depa ener een eee reer ae renee pent eer ren rare rer er rrr ter ree tre rrr rr rer 220 Ps ig tr lees MES a a rec cas carn au tik uN Gunihts staid R 221 sM FA EEE r E E E E E E E E aati nRED 221 S1 Me Open ATL Mes FlESnnnanssaa iaa teats endian iediads 226 aA Ur EE e A E E E A T A E fais beau atisaln dis daaibiubeesindnias 229 KAFE SE E E E 229 eME S NS E E E E E E E E E eeneaaas 231 h FETE Da EN gM aaa cg sbsivaclenn phospcindg bos saatin ppotvenees wabiuaaean punsNeRNeaeeaAS 231 s13 File Epro NON oP ee aieiaa T 232 el File EOT NEX scorre N 233 5 1 10 Fie Execute Baich Command Files sccsicsscxeestssiorien saatnassiriainca ienusens 235 aLi Eile Edi Balh LVM FIE abrinesnuiuiannonniai anaana aa 235 5 1 12 File View Log from Last Executed Batch Command File ccccsceeeeeeseees 235 5 1 13 File Print Print Preview Print Setup sccsisccscccdanazscscssnateonccinntatenduineteancanns 236 Siia Fie Pnt aD sear UU NAN Ssa NAA NON 236 5 1 15 File Send All Sort Summaries to PowerPoint cssceeeeeeeeeeeeeeeeeeeeeeeeeeesaes 236 9 1 16 Fle SCG Filg mesca 236 SA Fie E aina a A aa 236 TEAS U E 7 18 EA P T E T A E T E errr N E er terry cena A mir mre nit 237 Te FEJ UMO Pi gat ea E memiraeulds 237 B22 Egit Reload Channel from Filemoni 237 5 2 3 Edit Previous Channel in History Next Channel in History ccceeeeseeeeee 237 RAE E E E E A E E E A E E E te 237 Gal List of view Merti C
303. ny given time When a file is first loaded Offline Sorter Software will in general and for single electrode data create an Offline Sorter Channel for every raw channel in the data file The Channel Parameters view see Section 3 2 3 Channel Parameters View on page 30 shows the Source id and the raw channel number which together uniquely identify the raw channel for each Offline Sorter Channel in the column labeled Raw Src Ch see screenshot in next section Note An Offline Sorter Channel can be single electrode sterotrode or tetrode in which case it can correspond to 1 2 or 4 raw channels Note For most file formats checking Remove empty spike channels or Disable loading continuous data channels checkboxes in the Data Import Options General tab will prevent Offline Sorter Software from creating Offline Sorter Channels for certain raw channels from the data file Offline Sorter Software will automatically map raw channels to Offline Sorter Channels using a straightforward scheme but the user can control this mapping using the Channel Map view See Section 3 2 13 Channel Map View on page 37 Using this view the user can also re map and convert single electrode raw channels into stereotrode tetrode Offline Sorter Channels As stated above immediately after a data file is loaded each Offline Sorter Channel will correspond to 1 raw channel or 2 or 4 raw channels for stereotrode or tetrode data However each Offli
304. o F The Pseudo F statistic is closely related to the J3 statistic It is essentially J3 that has been adjusted for the number of waveforms and the number of units Pseudo F N g g 1 J3 where N is the total number of waveforms and g is the total number of units 308 Offline Sorter Offline Spike Sorting Software Sorting Quality Statistics 6 6 4 Davies Bouldin The Davies Bouldin validity metric is calculated as 1 d G d G DB NEM l di j where d i j is the Euclidean distance between the centroids of unit i and unit j and d i is the average distance of each point in unit i to the centroid of unit i The sum is over all N units and the max is taken over all units j i Because the inter cluster distance d i j is in the denominator the Davies Bouldin validity statistic takes on small values for well separated unit clusters The Davies Bouldin validity statistic is a non parametric measure 6 6 5 Dunn The Dunn validity metric is calculated as Dunn min min S aa where d i j is the Euclidean distance between the centroids of unit i and unit j and d k is the average distance of each point in unit k to the centroid of unit k The outermost min is taken over all units i the inner min is taken over all j i and the max is taken over all units k The Dunn validity index assumes a maximum value for clusters that are compact and well separated 6 6 6 Per Unit Sort Quality Metrics The abo
305. o a hand Mov ing the mouse up moves the eyepoint farther away and moving it down moves the eyepoint nearer TIP Manipulating the Eyepoint To get accustomed to manipulating the eyepoint think of the hand grabbing onto and turning the point clouds By default the eyepoint motion is constrained to not allow upside down eyepoints which can be confusing This constraint can be relaxed by unchecking the Constrain view rotation so that Y axis is always upwards checkbox in the Tools Options Display tab To animate the eyepoint rotation The 3D Clusters view supports a mode of operation that enables the eye point for the 3D view to be continuously animated This can be controlled by using the Animate the 3D rotation after left mouse release option in the Tools Options Display tab When this option is enabled the eyepoint con tinues to move in whatever direction it was going when the mouse button is released This is as if the left mouse button throws the eyepoint into an orbit around the look at point This option can quickly be toggled on or off by press ing the A key while the 3D Clusters view has focus The Look At Point the point of rotation in 3D Wherever the eyepoint is positioned the direction of gaze is always adjusted so that the eye is looking at a certain point in space This Look At point is marked on the 3D Clusters view with a small blue by default axis icon 66 Offline Sorter Of
306. o define a time range for the waveforms to export using the Export Only Waveforms in the Time Range checkbox Similarly one exports only the waveforms sorted into a particular unit by selecting the Only Unit radio button Select What to Export for Each Waveform This enables one to select what items appear in each column for each exported row of data The Available list box on the left shows all the items that can be exported and the Chosen list box on the right shows those that have been selected for export Click on one or more items to highlight them and move items back and forth between the list boxes by then pressing the Add gt gt or lt lt Remove buttons Use other convenience buttons to add all items remove all items or reset the chosen items to the factory defaults The order that the items appear in the Chosen list box controls the order that they appear in the output file You can change the order by selecting one or more items in the Chosen list box and pressing the Move Up or Move Down butions Offline Sorter Software remembers the chosen items and their order so that the next time information is exported the same items are chosen automatically Besides the obvious Channel Unit and Timestamp items you can export the value of any of the Features Available for Sorting for each waveform In addition to or instead of the exported feature values Offline Sorter Software can additionally export the raw A D values associated w
307. o two waveforms The Detector settings specify the quantity used in the threshold crossing algorithm for spike detection See Section 3 11 Waveform Detection View on page 74 By default Offline Sorter Software uses the Raw Signal amplitude If either Energy of the Signal or Nonlinear Energy of the Signal is selected the Energy Window Width can be specified To set the waveform detection parameters Select the Detector and Waveform Options button in the Timeline view Timeline _ l A Cont Ch 156 SPKC28 Showing a Control Displays Center Selected Zoom 1 06 2 50 mv Thr 10 43 iw P Detect Wavefor Options Detect Low cut Filter Bessel x 4 pole x Cutoff Freg 250 Hz Scroll Range 10 0000 0 0000 Zoom Out Zoor 120 Offline Sorter Offline Spike Sorting Software Waveform Acquisition From Continuously Recorded Data The Waveform Detection Options window opens Waveform Detection Options Extracted Waveforms Samples Waveform Length microseconds 32 Prethreshold Period microseconds 200 x 8 Dead Time microseconds 800 m 32 Note that OfflineSorter does not support different waveform lengths for different channels If you extract waveforms and then change the waveform length all the extracted waveforms will be erased For stereotrode tetrode data allow an independent threshold for each electrode Detector Detector Raw Signal Ad
308. oa New PLX File 206 Exporting toa New PL2 File 206 Exporting toa NEX File 206 Exporting Information 206 Exporting Per Waveform Data 207 Exporting Per Channel Data 209 Exporting Per Unit Data 211 Saving Template Files 212 Saving Templates 212 Sort Using Saved Templates 214 Saving and Restoring Scan Results 214 xii 157 158 160 163 165 168 171 173 175 176 178 182 184 186 190 199 199 199 205 Offline Sorter Offline Spike Sorting Software Offline Sorter Offline Spike Sorting Software Chapter 5 Menu Options 217 File Menu 220 File Open 221 File Import 221 Alpha Omega 222 CED Spike 2 222 Cyberkinetics 222 Data Translations 222 Datawave 222 MultiChannel Systems 223 Neuralynx 223 NeuroExplorer 223 Neuroshare Native 223 Panasonic MED 224 R C Electronics 224 Generic Binary Continuous File Import 224 Generic Neuroshare Import 226 File Open Multiple Files 226 File Close 229 File Save 229 File Save As 231 File Export to New PLX 231 File Export to New PL2 232 File Export to NEX 233 File Execute Batch Command File 235 File Edit Batch Command File 235 File View Log from Last Executed Batch Command File 235 File Print Print Preview Print Setup 236 File Print All Sort Summaries 236 File Send All Sort Summaries to PowerPoint 236 File lt Recent Files gt 236 File Exit 236 EditMenu 237 Edit Undo Redo 237 Edit
309. oducts The Products Licensed section tells which Plexon products are currently unlocked by the licensing key Note Offline Sorter Version 4 x is licensed separately from previous versions Contact Plexon for a software upgrade if needed Click Test the Key Again to re interrogate the key and re display the information The Unlock Additional Program and Features section applies to remote upgrades If a remote upgrade applies Plexon provides instructions on how to use this feature Multiple Plexon licensing keys can be installed on the computer Offline Sorter Software can find and use a license on any of the keys attached to the computer If Plexon software detects multiple Plexon keys on the computer additional Next Key gt gt and lt lt Previous Key buttons appear in the license Offline Sorter Offline Spike Sorting Software Demo Mode window These buttons can be used to step through the keys to examine each key individually 1 4 Demo Mode If a valid Plexon licensing key has not been connected to the computer Offline Sorter Software opens a startup message box to indicate that it is running in Demo mode The system provides all Offline Sorter features in Demo mode except the file saving batch processing and Export functionality 1 5 Off line Sorter Technical Support and Updates 1 5 1 Offline Sorter Technical Support If you need assistance with any aspect of Offline Sorter operations please contact Plexon su
310. of statistical tests on the separation of selected unit clusters in 2D and 3D feature space for the current channel The page is blank if a channel is not loaded For more information see Section 4 7 2 1 Info Grid Views Statistics on page 200 3 2 9 PCA View The PCA view displays the eigenvectors and eigenvalues for the waveforms in the current channel The first 8 eigenvalues from the PCA appear at the top of the display The eigenvector corresponding to each of those eigenvalues appears in a column directly underneath the eigenvalue This is a grid based view that numerically shows the same information displayed in graphical form in the Section 3 15 PCA Results View on page 88 3 2 10 Scan View The Scan view is a data exploration tool that allows multiple automatic and semi automatic sortings on a channel while stepping the value of a sorting parameter over a range of values This provides a feeling for how the sorting 36 Offline Sorter Offline Spike Sorting Software Info Grid Views parameters affect the sorting quality For more information see Section 4 5 12 Parameter Scans on page 179 3 2 11 Templates View The Templates view displays a numerical representation of the template for each unit These templates can be copied and pasted into other analysis programs Note that the templates will not be displayed if Offline Sorter Software is running in Demo mode 3 2 12 Adaptive Templates History View Th
311. of the screen e Show Cluster Quality shows the selected Sort Quality metric changes at the top of the display as described earlier e Show Ellipses Ellipsoids shows the ellipses for units ellipsoids for the 3D Clusters View as described earlier e X Range Y Range expands contracts or shifts the viewing range as described in Zooming and Panning earlier e Reset Range And Zoom resets the zooming and panning for the view to values calculated so that all the points are visible 3 7 2 12 2D Clusters View Details Showing Dots or a Density Histogram In response to the selection made with the right click menu the 2D Clusters view can display the feature space points as individual dots a density histogram or both Displaying as a density histogram builds a 2 dimensional histogram of the points and maps the histogram bin contents to a gray scale The information that appears in the 2D view is the same information that appears in the Surface view in 3D format for more information see Section 3 8 Surface View on page 69 To display the feature space points as dots the default From the 2D Clusters shortcut menu ALT right click select Show Dots Version 4 0 61 3 Views and Displays 2D Clusters 40x 2D Clusters 3D 3D Clusters Surface Peak Histo To display the feature space points as a density histogram From the 2D Clusters shortcut menu ALT right click select S
312. old ie positive or negative the Same Side as Threshold radio button e Adjust Gains This button brings up the Adjust Gains Tool which allows for reconciling the voltage ranges between the continuous data and spikes See Section 4 2 4 Adjusting Gains on page 115 e Detect Now This button will perform the waveform extraction it is equivalent to pressing the Detect Waveforms button on the Timeline view If energy or nonlinear energy is selected as the signal to be thresholded in the Detector options menu the quantity is displayed in the Timeline view and the 122 Offline Sorter Offline Spike Sorting Software Waveform Acquisition From Continuously Recorded Data histogram of the quantity log axis is displayed in the Waveform Detection view Waveform Detection ESTE M ea le m il lh il M AN 4 2D Clusters j Waveform Detection 3D 3D Clusters Surface The threshold can then be adjusted to different energy nonlinear energy levels or histogram values 4 2 7 Extracting the Thresholded Waveforms Offline Sorter Software uses the following algorithm to extract the waveforms from continuously recorded signals If the threshold is positive or zero the program looks for the moment when the signal crosses the threshold in the positive direction find the first t such that signal t dt lt threshold and signal t gt threshold If the threshold is negative the program looks for the moment
313. on 4 0 45 3 Views and Displays e Add Unit Unit clusters in this view can be created by using the mouse pointer to cross waveform bundles see Section 4 5 3 Manual Clustering using the Waveform Crossing Method on page 153 e Delete Unit Deletes the currently selected unit e Add Wfs Adds the crossed waveforms to the currently selected unit e Remove Wfs Removes the crossed waveforms from the currently selected unit i e change them to unsorted e Invalidate Wfs Marks the crossed waveforms as being invalid so that they are ignored See Section 4 6 3 Cleaning the Selection on page 187 To control the temporal range and type of waveforms shown in the Waveforms view it is possible to use the scroll bar at the bottom of the Timeline View when the Control Displays check box is checked and the Waveform View parameter in the Control Grid The Draw Grid option in the Control Grid determines if a reference grid appears in the Waveforms view The Waveforms view will show the template waveform for each unit drawn as a single complementary colored waveform By default the Waveforms view will show the template waveforms for all units along with the sorting boxes and lines for all units if in Boxes or Lines sorting mode But by selecting Show Templates Boxes Lines only for Selected Units from the right click menu the Waveforms view can be made to show the templates and boxes and lines for only the selected u
314. on 4 0 229 5 Menu Options the unit assignments for spike records are modified to reflect the sorting that has been performed in Offline Sorter Software There are several options to control how the File Save mechanism works This mechanism is available in the File Save Behavior drop down list in the General tab of the Tools Options dialog box When files are saved using the File Save menu option or the toolbar Save button Offline Sorter Software provides control over saving files in terms of whether the data files are overwritten or backups are kept You can control this behavior using the File Save Behavior drop down list in the General tab of the Tools Options dialog box The options available are e Preserve Original prompt for new filename defaults to filename 0n This is the behavior available in older versions of Offline Sorter Software The original data file is never altered but Offline Sorter Software prompts for a new file name to write the sorting results into During the prompt a default file name appears that is constructed by appending numbers to the original filename until an unused filename has been made The latest sorting results are in the filename On file with the largest n e Overwrite Original backup to filename 0n before each save With each save Offline Sorter Software always overwrites the original file with the latest sorting results but it saves the previous version of the file to a backu
315. on all channels The result is then exported to a new PLX or PL2 file such that the invalidated waveforms are not saved Note that this batch command file must be run on a data file that contains spikes 6 10 3 ScanKMeans ofb Performs a 2D K Means Scan on all channels and saves the scan results to SCAN files The SCAN files can be loaded back into Offline Sorter Software to make the results available for inspection Note that this batch command file must be run on a data file that contains spikes 6 10 4 ScanValleySeeking ofb Performs a 3D Valley Seeking Scan on all channels and saves the scan results to SCAN files The SCAN files can be loaded back into Offline Sorter Software to make the results available for inspection Note that this batch command file must be run on a data file that contains spikes 6 10 5 FiltDetect ofb Performs low cut filtering and waveform detection on a continuous data file and exports the extracted spikes to a PLX file Note that this batch command file must be run on a data file that contains continuous data 6 10 6 SaveToNex ofb Performs low cut filtering and waveform detection on a continuous data file sorting and artifact invalidation then exports everything to a NEX file Note that the InvalidateArtifactsAfter command is used in order to perform the artifact invalidation after the waveform extraction has been completed Note that this batch command file must be run on a data file that contains cont
316. on displays the following dialog box Excel O Matlab file Which Channels Delimiter to use between values Current Channel only Comma x D All Channels into one file X7 Export a Row of Column Headers What to Export for Each Unit Available Chosen RMSE File Channel Raw Channel Name Channel Name Raw Sort Method Used Small ISI Center Xx 2D Center Y 2D Ellipse Angle 2D Sigma x 2D Sigma Y 2D Center x 3D Center Y 3D Center Z 3D Sigma x 3D x V Append Template Waveform Data in units of Also Append Template Std Dev Data E PlexonD ata Plexon plx testwaveforms sorted txt Unit Number Add gt gt Number of Waveforms Add All lt lt Remove Remove All Move Down Move Up microVolts 0 ADC Counts What File to Store the Exported Data in Browse Append if existing Cancel To perform an export 1 Select the Format for the exported information either text file MATLAB file if MATLAB is available on the computer or Microsoft Excel if available on the computer For text export choose a delimiter to use between values Delimiter choices include comma comma and space space or semicolon For Excel and text export check the Export a Row of Column Headers to get a header line of text that labels each of the columns This makes reading the exported file easier Select Which Channels to
317. on the Waveforms view for each unit all waveforms for a unit must intersect all of the line segments for a unit e Section 4 5 7 Manual Clustering using the Bands Method on page 168 e The user selects and adjusts both a template waveform and a corridor or band around the template on the Waveforms view for each unit all waveforms for a unit must remain within the band e Section 4 5 8 Semi Automatic Clustering using K Means on page 171 The user selects tentative cluster centers in feature space in the Clusters views and then runs the K Means algorithm which assigns points to clusters automatically e Section 4 5 9 Semi Automatic Clustering using Standard E M on page 175 144 Offline Sorter Offline Spike Sorting Software Spike Sorting e The user selects tentative cluster centers in feature space in the Clusters views and then runs the Standard E M algorithm which assigns points to clusters automatically e Section 4 5 10 Automatic Clustering using Valley Seeking Method on page 176 e The number of clusters and cluster membership is automatically determined via a Valley Seeking algorithm operating on nearest neighbor distances in feature space e Section 4 5 11 Automatic Clustering using T Distribution E M Method on page 178 e The number of clusters and cluster membership is automatically determined via an E M algorithm based on the T Distribution e Section 4 5 12 Parameter Sc
318. ons they have the same effect as the checkboxes 4 6 3 Cleaning the Selection Offline Sorter Software also provides the tools to remove waveforms from a selected unit that is to label the waveforms as unsorted For example it may be desired to remove some of the outlying waveforms from this unit 4 6 3 1 Automatic Outlier Removal Tool The Tools Remove Outliers menu selection provides a tool that automatically removes outliers from clusters based on the Outlier Thr setting in the Control Grid This outlier removal works by considering the location of the points corresponding to the waveforms in the currently selected feature space If the 3D Clusters view appears when the tool is selected then 3D feature space is used otherwise 2D feature space is used The outlier removal tool automatically does the following for each unit e Calculates the centroid and sigmas along the principal axis in the 2D or 3D feature space e For each point calculates the Mahalanobis distance between the point and the centroid The units of Mahalanobis distance are standard deviations e If this distance is greater than the Outlier Thr setting it sets the waveform point to unsorted Version 4 0 187 4 Spike Sorting Step by Step 4 6 3 2 Manual Waveform Removal You can also manually remove any waveform not just outliers from a unit This feature is easier to use if the Waveforms view shows only the waveforms of a particular unit
319. ontinuous data SaveNexProcessedCont set to 1 to have ExportToNEX save the unsampled and filtered version of the continuous data set to 0 to save the raw continuous data SaveNex Waveforms set to 1 to have ExportToNEX save waveforms set to 0 to ignore them and just save the timestamps SaveNexUnsorted set to 1 to have ExportToNEX save unsorted waveforms set to 0 to ignore them SaveNexUnitTemplates set to 1 to have ExportToNEX save the unit templates to a waveform variable in the exported NEX file 320 Offline Sorter Offline Spike Sorting Software Batch File Processing e ExportWaveformsFileType ExportChannelsFileType ExportUnitsFileType set to either txt mat or xls to have the ForEachFile ExportWave forms Channels UnitsInfo export functions write to text files a MATLAB file or an Excel spreadsheet respectively 6 9 1 6 Process The Process command processes all queued files Resets the queued files list Syntax Process 6 9 2 Example Batch Command File Here are the contents of example batch file ofsbatch ofb ForEachChannel ValleySeek ForEachChannel SortSummaryToPP ForEachChannel LogStats2d ForEachChannel LogSiats3d ForEachFile ExportToNex Set Featurex 0 Set FeatureY 1 Set ParzenRadiusMult 1 0 File C PlexonData Plexon plx chan9threeunits3 pIx File C PlexonData Plexon plx ev024 plx Process Set ParzenRaadiusMult 2 0 File C PlexonData Plexon plx chan9threeunits3 plx
320. ools Options 270 Tools Data Import Options 271 Offline Sorter Offline Spike Sorting Software Offline Sorter Offline Spike Sorting Software Window Menu 272 Run Menu 272 Help Menu 273 Help Topics 273 Quick Key Reference 273 Quick Batch Reference 274 About Offline Sorter 274 Chapter 6 Reference Topics 275 Details of Channels and Sources 277 Raw Channels and Sources 277 Offline Sorter Channels 278 Offline Sorter Channels and Sources 278 Viewing Corresponding Channels 282 Channel Mapping 286 Stereotrode and Tetrode Channel Mapping 288 Converting Data Type 288 Loading a Channel Map from a File 290 Saving a Channel Map to a File 291 Stereotrode and Tetrode Data 291 Loading Stereotrode or Tetrode Spikes 291 Extracting Stereotrode or Tetrode Spikes from Continuous Data 292 Sorting Stereotrode and Tetrode Spike Waveforms 293 Disabling Electrodes 294 Features Available for Sorting 294 Principal Components 297 Voltage at Specific Slices in Time 299 Details of the Sorting Algorithms 299 Feature Space Used for Sorting 299 Template Algorithm 300 MAD Calculation 301 Adaptive Templates Algorithm 302 K Means Algorithm 303 Standard E M Algorithm 303 Valley Seeking Algorithm 304 T Distribution E M Algorithm 305 Partial Sorts 306 Sorting Quality Statistics 306 MANOVA F and p values 307 Some details on the MANOVA calculation 307 J3 308 Pseudo F 308 Davies Bouldin 309 Dunn 309 Per Unit Sort Quality Metrics 309 Thresholding A
321. or the channel being loaded i Sorting Results will Load from SCAN File Note that the sorting results for this channel will be automatically loaded from a companion SCAN file that was saved previously The automatically loading of companion SCAN files can be disabled in the Tools Import Options General options page If the companion SCAN files contain sorting results that are no longer of interest delete them for ALL channels by pressing this button This will then load the original sorting contained in the data file Delete All Companion SCAN Files for this Data File 1 Do not show this dialog again Do NOT load from the SCAN File this time The dialog contains a button to delete all SCAN files that fit the above format and match the current filename This function is also available from the Sort Delete All Companion SORT Files menu If desired loading the scan result from the SCAN file can be skipped for this channel this one time next time the channel is loaded it will again attempt to load the companion SORT file Checking the Do not show this dialog again checkbox is equivalent to unchecking the Warn when loading sorting results from a SCAN file checkbox discussed above and will cause companion SCAN files to be loaded always As covered in Section 6 9 Batch File Processing on page 313 a sorting parameter scan can be done from a batch command fi
322. p file Offline Sorter Software constructs the backup file name by appending numbers to the original file name e Overwrite Original backup to filename On on first save only This item is the same as above except that Offline Sorter Software makes the backup only on the first save That is the first time that a file is saved Offline Sorter Software saves a backup of the original to a backup file whose filename it constructs by appending numbers to the original file name Offline Sorter Software overwrites the original file Subsequent saves overwrite the original file but they do not save backups With this option the filename On backup file contains the data originally read into Offline Sorter Software no matter how many times the file is saved e Overwrite Original backup to filename 0n and update each save On each save Offline Sorter Software overwrites the original file and the backup file The backup file contents are always one save behind the original file e Overwrite Original do not backup This item just overwrites the original file on each save and doesn t make backups Plexon does not recommend this option unless the data files are backed up elsewhere 230 Offline Sorter Offline Spike Sorting Software File Menu 5 1 6 File Save As Note 5 1 7 File Export to New PLX The File Save As function is the same as the File Save function except that the user explicitly specifies a n
323. p the rest will be invalidated Keep Waveforms that Occur between Event and Event B e g Start and Stop Z Event Start v Event B Stop x Time Offset Before or After Event A for Start of Interval 0 Time Offset Before or After Event A for End of Interval 0 0 0 000000 29 481725 Which Channels Current Channel only E Create Time Segments All Channels in Spike Source Erase existing SPK Chs 1 64 First select the desired action in the Keep Waveforms that droplist The rest of the controls on the dialog either enable or disable as is appropriate for the droplist selection The Keep Waveforms that droplist selections are Occur between Event A and Event B e g Start and Stop This selection enables both the Event A and Event B droplists and shows a list of all the event names that occur in the data file Pick the Event A that marks the beginning of the desired waveforms and pick the Event B that marks the end The waveforms that are not between Event A and Event B are invalidated Occur within a time span around Event A This selection enables the Event A droplist as well as the time offset edit boxes Select the Event in the Event A droplist and enter the number of milliseconds before and after the event for which waveforms are to be kept Occur outside of a time span around Event A This selection is the logical opposite of the previous option All waveforms are kep
324. p gains are set to in Offline Sorter Software and so the per channel gains are the total gains For a variety of reasons gains can sometimes be mis represented in data files When this happens voltages that Offline Sorter Software reports may be off by some constant factor In many cases this is not really a problem as all the waveforms shapes are still correctly represented and spike sorting depends only on relative voltage differences not absolute voltages The File Summary in Info Grid Views shows an overview of the gain situation for a file and the Channel Parameters view shows the individual gains for all channels The individual gains can be adjusted using the Channel Parameters View and in general these gain changes will affect the interpretation of the currently loaded data and will be saved with an Export to PLX operation There is also a Tools Adjust Gains dialog that offers a convenient way to see the detailed gain situation for the current channel This dialog can also be brought up from the right click menu in the Channel Parameters View Version 4 0 115 4 Spike Sorting Step by Step r Adjust Gains bed a Channel He FP12 0 Waveforms 8575 Continuous Samples Spikes Full Scale Voltage at Probe mV 2 500 lt lt lt Peak Voltage at ADC Input mv 2500 000 A D Resolution bits 16 Voltage Resolution at Probe mV bit 0 000076 Optimize Preamp Gain 1 Total G
325. pera tions that are not saved in TPL files include e any operation that invalidates waveforms e g removing outliers e multiple applications of alignments or sorting operations Only the last operation is saved to the TPL file e manually adding or removing waveforms from clusters 4 8 4 2 Sort Using Saved Templates You can sort both continuous data files and spike waveform data files using templates saved in TPL files To sort a file using saved templates first open the continuously digitized data file or open the spike waveform data file see Section 4 2 1 Opening the Digitized Data Files on page 110 To select the tpl template file Select Sort Sort Using TPL file menu command The templates tolerances contours feature selections extraction and alignment parameters and sort widths that were saved for each channel in the TPL file are then used by Offline Sorter Software to sort the current data file For continuously digitized data files threshold low cut filter detector type as described in Section 4 2 6 Specifying the Waveform Detection Options on page 120 settings are also saved in the TPL file for each channel and applied to the new file to extract the waveforms for sorting When a channel is sorted using a saved TPL file the order of operations 1s 1 extract the waveforms using the saved parameters for continuous data channels only 2 perform alignment on the spikes if indicated by the sa
326. performing this operation view the probabilities and F statistics reports with caution The Only align waveforms assigned to the currently selected unit option is similar to the previous option in that it only performs the alignment on the current primary selected unit The For stereotrode tetrode data align waveforms in each electrode independently checkbox is only relevant when working with stereotrode tetrode data When unchecked the concatenated waveform See Section 6 3 Stereotrode and Tetrode Data on page 291 is aligned as a whole the min or max of the entire concatenated waveform is found and the concatenated waveform is shifted as a whole When checked the alignment is instead performed per electrode For example for tetrode data four independent alignments are performed one for each electrode Note that the waveform in each electrode can be shifted a different amount Version 4 0 141 4 Spike Sorting Step by Step The Adjust Timestamps checkbox controls whether or not the timestamp of the waveform is shifted by the amount the waveform is shifted The Maximum Alignment Shift adjustment enables one to put a cap on the number of clock ticks that any waveform is shifted It is seldom desirable to shift a waveform by more than a few clock ticks because zeros are added onto the end of the waveform to backfill the shifted amount Waveforms that would be shifted by more than the specified number of clock ticks are
327. pikes for Overlaps Analysis it is best to only sort the waveforms that are clearly and obviously members of a unit leave any outliers or questionable waveforms unsorted Any sorting method may be used for the initial sort but in the next step the waveforms will be re sorted using the unit templates found during this step Remember that the goal is to get an accurate unit template for each of the true units and leave the potential overlapping Version 4 0 195 4 Spike Sorting Step by Step waveforms as unsorted so that the Overlaps Analysis algorithm can potentially recognize them as overlaps and rescue them It is best if low amplitude noise waveforms have not been sorted into a unit It is recommended that you invalidate these or re threshold so that these low amplitude waveforms are not present To rescue overlapping waveforms at least two units must be sorted Resort the Units using Template Sorting The Overlaps Analysis procedure relies on combining offset versions of unit templates and it will implicitly sort units and their overlaps using the Template Sorting algorithm Thus the algorithm implicitly assumes that all waveforms have already been sorted according to whether they match the unit templates Or perhaps more accurately it is assumed that unsorted waveforms do not match any unit template to within tolerance Note that the role of the Tolerance is two fold first since it is applied during the Templa
328. plate Sorting In analogy to the Perform Automatic Sorting dialog selecting Sort Perform Template Sorting from the main menu will display this dialog r Perform Template Sorting Which Channels pumcosesisvscessteosasbeess tossesbecsabeosenssaet All Channels in Spike Source SPK Chs 1 64 Template Sorting Method and Parameters v Apply Changes Immediately Standard Template Sorting Override Template Tolerance ta 70 for All Units Adaptive Template Sorting Weight 0 01 Update Threshold 20 Current Channel 0K tance which provides a convenient place to explore and apply template based sorting methods to spikes The Template Sorting will always use or start with the current Templates defined for each existing unit in a channel so units must already be defined before running this tool When applying standard Template Sorting the user can override the per Unit template tolerance to a specific value When applying Adaptive Template Sorting see Section 6 5 4 Adaptive Templates Algorithm on page 302 you can control the adaptive template algorithm Weight and Update Threshold parameters Checking the Apply Changes Immediately Version 4 0 253 5 Menu Options checkbox will re apply the Template Sorting as soon as the template tolerance or adaptive template parameters are changed in the dialog As in the Perform Automatic Sorting tool the user can sel
329. pport at 1 214 369 4957 or support plexon com 1 5 2 Offline Sorter Updates The latest version of Offline Sorter Software may be downloaded from the Plexon website http www plexon com The standard Offline Sorter setup programs are OFSv4w7Setup exe for 32 bit machines and OFSx64v4w7Setup exe for 64 bit machines To install the latest version of Offline Sorter Software download the program to the PC desktop and run it Version 4 0 1 Getting Started 6 Offline Sorter Offline Spike Sorting Software Plexon Inc Chapter 2 System Overview 21 Oline Sorner USGS naccnissiiniisnas saath 2 2 Offline Sorter NOUS cusses cnnmisnetsusipenuinindaunune 23 SOU Tecniques iinne a 2 4 Offline Sarier OUTS aenn aaa A41 Printing PROBUS sissoo iassuersiinuerinnnsisi 24 2 Saving POSUNG niiair 2 4 3 Reproducing ABSUNS ssscisiccscsictrcacsssterarsiiwiven CRB Ti ite 3 8 aetrrereeente err erent ere ree rr ere er ener rene ere ee 2 5 1 Spike Waveform Segments ccceeeeees ce CCUM Eiaeaen Eii 2S LUCCA u iaadeaentaasesenies And Fast ahd SIO onsi Toa Cian ne cer rtrar Pes tere rer re PAS SUIS ccsaeattecacte tere tcaneda de coaueatty tandem ceedeanteen 2 6 Offline Sorter Screen Layout cccccceeceeeeseeeeees yl NOONE annaa 202 platus AF omainen AA E o T E O A N E Version 4 0 2 System Overview 2 1 Offline Sorter Uses Offline Sorter Software can be used to Inspect the data Offline Sorter
330. r SpanEvent interval filter Filter will invalidate waveforms outside of the time interval time of SpanEvent SpanEventStartOffset to time of SpanEvent SpanEventEndOffset TimeStart TimeEnd defines the time interval in msec to use for the KeepWfsBetweenTimes and InvalidateWaveformsBetweenTimes interval filters SortType sets the sorting type to use for the ForEachChannel Sort command The argument values are ValleySeek TDist ScanK Means ScanStdEM ScanValleySeek ScanTDist SortDim either 2 or 3 to select the dimension of the feature space used for sorting ParzenRadiusMult sets the parzen radius multiplier for Valley Seeking sorting to the specified value DOFMiult sets the degrees of freedom multiplier for T Distribution E M sorting to the specified value OutlierThreshold sets the outlier threshold to the specified value RefractoryPeriod sets the refractory period to the specified value for the RemoveShortISI channel command WaveformLimit sets the limit for the number of waveforms used in the Valley Seeking sorting For more details see Section 4 5 10 Automatic Clustering using Valley Seeking Method on page 176 Version 4 0 319 6 Reference Topics FeatureX FeatureY FeatureZ set the feature for each dimension of feature space See the following table for numerical values for the features Slice1 Slice2 Slice3 Slice4 set the slice position in ticks for the Slic
331. r the Only Segment checkbox in the Control Grid is checked then the Clusters and Waveforms views will show only waveforms inside the currently selected time segment 4 6 2 Waveform Selection Options Use the Operations Involving Waveforms section in the Tools Options General dialog to determine which waveforms are included in waveform operations such as sorting selecting waveforms adding waveforms removing waveforms etc There are three options either use all waveforms or use only the currently visible waveforms or use only the unsorted waveforms If the Use Only Currently Visible Waveforms checkbox is checked Offline Sorter Software restricts operations to affect only those waveforms that are currently visible in the Waveforms view or Clusters views as appropriate For methods 186 Offline Sorter Offline Spike Sorting Software Unit Cleaning of selecting which waveforms are made visible see Section 4 6 1 Controlling Waveforms Displayed on page 184 If the Use Only Unsorted Waveforms checkbox is checked Offline Sorter Software restricts operations to affect only those waveforms that are currently unsorted One must use either the Use Only Currently Visible Waveforms option or the Use Only Unsorted Waveforms option both options cannot be used together You can also use the Waveforms Use Only Currently Visible Waveforms or Waveforms Use Only Unsorted Waveforms menu options or the corresponding toolbar butt
332. r variable names that hold the unsorted waveform timestamps have a U appended to denote Unsorted waveforms 5 1 10 File Execute Batch Command File Offline Sorter Software provides the ability to execute a subset of its commands via a script By convention batch file scripts for Offline Sorter Software have the file extension ofb but any file extension is legal If a text file is selected with the File Execute Batch Command File menu item it results in that file being read and interpreted as batch commands See Section 6 9 Batch File Processing on page 313 for the details of the batch file commands 5 1 11 File Edit Batch Command File For convenience Offline Sorter Software provides the ability to edit a batch command file with the File Edit Batch Command File menu selection This command will remember the last used batch command file and offer it as a suggestion in the File Open dialog After a file is selected Offline Sorter Software will launch a desired editor notepad exe by default to edit the selected batch command file To change the editor that is launched by this command select Tools Options General and select the editor executable in the Editor to use for Batch Command Files section 5 1 12 File View Log from Last Executed Batch Command File When a command file executes Offline Sorter Software shows the progress in a dialog and also writes a log file with a log extension appended to the ori
333. rawing a white rectangle around it F Control Displays Center Selected Zoom 10E 1 50 mY Thr 2 12 1 181 p Option Up Sampling None Frequency N A Low cut Filter None Scroll Range 9 500 0 2500 Zoom Out Zoomin lt The selected waveform is then viewable in the Waveform Inspection Window 4 7 1 2 Selecting Waveforms in the Clusters Views 3D Clusters Display 16Ch E4 Waveform Insp Run Yalley Seeking Delete Selected Unit To select a waveform left click on the waveform s dot in the Clusters view hold down the ALT key before left clicking if the 3D Clusters view is being used The selected waveform is then viewable in the Waveform Inspection window as shown above 4 7 1 3 Comparing the Waveform with the Templates The waveform selected in the Timeline view or the Clusters views drawn in white in the Waveform Inspection window is plotted against the average Version 4 0 199 4 Spike Sorting Step by Step waveform template of each sorted unit drawn in color in the Waveform Inspection window Info BE 3D Clusters gs Inspection The degree to which the selected waveform matches each of the sorted unit Templates is given by the sum of squared difference between the Templates and the waveform shown in the upper right of each plot The Unit with the minimum sum of squared difference with the selected waveform is denoted with the symbol best match in the upper portion o
334. rd Run Menu Options Personalized Menus and Toolbars Aways show full menus V Show full menus after a short delay r Reset menu and toolbar usage data Tool Bars Large icons Show Text Under Icons A 22 Offline Sorter Offline Spike Sorting Software Personalized Menus and Toolbars This area contains two check boxes and a button The application will hide infrequently used menu items but you may customize the display of menu items using these two checkboxes If you check the Always show full menus checkbox the application will always show full menus and the Show full menus after a short delay checkbox will be disabled If you do not check the Always show full menus checkbox you will have the option of checking or clearing the Show full menus after a short delay checkbox The Reset menu and toolbar usage data button will allow you to delete the record of commands you have used in the application and restore the default set of visible commands to the menus and toolbars A confirmation dialog displays Tool Bars If you check the Large icons checkbox the application will use large icons on the main application toolbars If you check the Show Text Under Icons checkbox you will also see text labels displayed under the toolbar icons Version 4 0 A 23 Appendix A Navigating the Plexon UI A 24 Offline Sorter Offline Spike Sorting Software Plexon Inc Index Symbols NEX fi
335. re specified in seconds from the beginning of the file A special value of 1 for the channel number is interpreted as meaning apply to all channels e Occur within a Segment With this selection an existing Segment must be selected from the droplist All waveforms that occur outside of the time range associated with the selected Time Segment are invalidated e Occur outside of a Segment With this selection an existing Segment must be selected from the droplist All waveforms that occur within the time range associated with the selected Time Segment are invalidated This is the inverse of the above option You can choose to invalidate the waveforms from just the current channel or from all channels in a selected Source You can make this selection by using the radio buttons under Which Channels in the dialog box If Offline Sorter Software reads the ranges in from a text file it determines the channels affected based on the contents of the Ranges file If the Create Time Segments checkbox is checked the Interval Selection Tool will create time segments that correspond to the intervals for which it kept or invalidated waveforms By default the new time segments will be added onto the list of existing time segments but checking the Erase existing checkbox will erase any existing time segments before creating the new time segments that correspond to the invalidation intervals As soon as the OK button is pressed the filtering
336. req edit box displays the cut off frequency of the continuous data low cut filter Changing this value will enable the Apply button Pressing the Apply button will apply the current low cut filter as defined by the filter type number of poles and cutoff frequency to the continuous data in the channel Scroll bar Zoom Out Zoom In Use these controls to change the limits of the scroll range for the Timeline view Scroll range tThe scroll window length and start time appear to the left of the scroll bar the end time appears to the right 50 Offline Sorter Offline Spike Sorting Software Timeline View 3 6 2 Other Features of the Timeline View Right clicking on a waveform brings up a menu that enables one to Mark Waveform Valid if the selected waveform is invalid mark it valid Mark Waveform Invalid if the selected waveform is valid invalidate it Unsort Waveform remove the waveform from its currently assigned unit making it unsorted Add Waveform to Selected Unit assign the selected waveform to the current unit Add Waveform to New Unit create a new unit and assign the selected waveform to that unit Use Waveform as Template for New Unit and Sort use the selected waveform as a template to define a new unit using Templates sorting See Section 4 5 4 Semi Automatic Clustering using Templates on page 157 Create Single Time Segment Use this to replace all existing time segments with a new time segme
337. rlapping windows Offline Sorter Offline Spike Sorting Software Close Button The Close button closes the window Plexon software applications often display several windows simultaneously These windows may be resized by using standard resizing methods and you may also reposition windows by dragging and dropping and by using Docking Stickers The illustration below shows the repositioning of a window denoted by the blue transparent rectangle and various Docking Stickers with red circles around them These Docking Stickers allow you to dock the window being moved in one of several ways described below iiiad irt iHi You may position a window by floating it docking it at the desired docking sticker at a window edge or in a tab Floating a Window You may drag a window by the Caption Bar near the center of the screen and release it causing the window to float Holding down the CTRL key while dragging will always float the window Double clicking on the Caption Bar will also float a window Note that the size and position of the floating window is remembered Rolled up Windows Floating windows may be enabled for roll up by pressing the Auto Hide button The window will roll up when the focus is changed to a different window The first illustration below shows a floating window before rolling it up The second illustration below shows the rolled up window after the focus has changed Version 4 0
338. rogress bar will appear in the middle of the status bar On the right side are text fields that change based on the state of the program such as the approximate amount of memory that is still available to use Of particular importance are the time voltage and feature space labels that display the values corresponding to the mouse position within some of the views Version 4 0 17 2 System Overview 2 6 3 Views The bulk of the user interface of Offline Sorter Software consists of many independent views or windows that can be opened docked floated and placed into tabs See Appendix A Navigating the Plexon User Interface Each available view within Offline Sorter Software has a corresponding menu item under the View main menu entry and many of the common views also have a corresponding toolbar button Some of the views within Offline Sorter Software have associated with them their own Toolbars The presence of the per View Toolbars is indicated by the small triangle in the title bar For example the title bar for Waveforms looks like this Pressing the triangle will toggle showing or hiding the per View toolbar When shown the per View toolbar can include both icons and text 2 K x Fes i Add Unit Delete Unit Add Wfs Remove Wfs Invalidate Wfs or just the icons Showing the text can be turned on or off by right clicking on the toolbar and selecting the Text Below Icons item from the menu
339. rol Grid or the Waveform display Slice 3 w k the waveform voltage at time t k e Slice 4 The waveform height at a selected position in time You can select the position in the Control Grid or the Waveform display Slice 4 w the waveform voltage at time t l e Peak The maximum voltage amplitude across the entire waveform length Peak max w t e Valley The minimum voltage amplitude across the entire waveform length Valley min w t e Peak Valley The difference between the maximum and minimum waveform amplitude Peak Valley max w t min w t Version 4 0 295 6 Reference Topics Peak Valley FWHM The Full Width at Half Maximum for the peak or valley of the waveform expressed in milliseconds The exact points where the waveform crosses the half max value are obtained by interpolation Peak Valley Tick The clock tick as measured from the beginning of the captured waveform at which the peak or valley occurs No interpolation or waveform fitting is done so these values are quantized to integer values Area The total integrated area underneath the waveform Area Zabs v t Energy The waveform energy as it is calculated for spike detection See Waveform Detection View on page 74 1 Energy eww v t Sqrt Energy The square root of the above mentioned waveform energy Non Linear Energy The non linear waveform energy as it is calculated for spike
340. roperly sorted Note that there is an underlying assumption that the waveforms produced from the firing units add linearly to form the overlapping waveform that is recorded in the continuous data The Overlaps Analysis is only suitable in the following situations e The spikes were created within Offline Sorter Software by running waveform detection on continuous data using a simple Raw Signal threshold This is required because in order to reconstruct the overlapping waveforms Offline Sorter Software needs access to the continuous data from outside of the spike window of the original overlapping waveform e The spikes have been sorted into at least two units using Template Sorting Units are required because the analysis is based on the unit templates of the current set of units Note that the original units could have been created by any supported sorting method but the overlaps analysis really only works well if the waveforms have been re sorted using the unit templates For convenience there is a facility for re sorting the waveforms using templates right on the Overlaps Analysis view window e Overlaps Analysis will only rescue unsorted waveforms It will not steal sorted waveforms from existing units 100 Offline Sorter Offline Spike Sorting Software Overlaps Analysis View 3 22 1 Overlaps Analysis Operation e Currently overlaps analysis is not supported for stereotrode or tetrode data This may
341. rrent Thresholds This setting uses the thresholds that are currently set for each channel You can view and edit the threshold settings for each channel in the Info Channel Parameters tab Version 4 0 249 5 Menu Options e Use a Constant Threshold Type a constant threshold value into one of the following boxes of ADC Range the percentage of the analog to digital converter ADC range indicated in the Timeline view see Section 3 6 Timeline View on page 48 microVolts voltage threshold in uV ADC Counts the threshold in raw analog to digital conversion counts Note Entering a value in any of the boxes will update the values in the other two boxes e Use Standard Deviations from Mean of Peak Heights Histogram Type the number of standard deviations sigmas from the mean to set the threshold at that distance from the mean of the peak height histogram A negative number sets the threshold to be less than the mean A positive number sets the threshold to be greater than the mean 5 To start the waveform detection process click Detect 5 5 15 Waveforms Align Select this menu item to align the spikes in a spike channel in time see Section 4 4 4 Waveform Alignment on page 138 This menu item is only enabled if there are spikes in the current channel 5 5 16 Waveforms Re Threshold This menu item has the same effect as the Tools Re Threshold Waveforms option
342. rsion 4 0 241 5 Menu Options Help Waveforms Sort Units Tools Window Run View Only Currently Selected Unit s View Only Unsorted Waveforms View Only Selected Time Segment Create Time Segments Use Only Currently Visible Waveforms Use Only Unsorted Waveforms Invalidate Waveforms Invalidate All Except Selected Waveforms Mark All Waveforms As Valid Invalidate All Waveforms in Selected Unit Perform Digital Referencing on Continuous Data Up sample Continuous Data Filter Continuous Data Detect Align Re Threshold Remove Short ISI Waveforms Auto Zoom Waveform Displays The sections that follow explain the contents of each Waveforms menu options 5 5 1 Waveforms View Only Currently Selected Unit s This is a toggle able menu item When it is selected checked the Waveforms view and optionally the Clusters views and the Timeline view will show only the waveforms from the currently selected unit Selecting this menu item is equivalent to either of the following e Selecting Sel Unit s from the Waveform View droplist in the Control Grid or e Pressing the _ button in the toolbar Whether this selection applies only to the Waveforms view or to the Clusters views and the Timeline view is determined by the setting of the Cluster Views and Timeline View droplists in the Control Grid if they are set to As Above meaning the setting for the Waveform View above
343. rt units using the current templates and tolerances To set the colors assigned to each of the sorted units select the Colors tab of the Tools Options dialog box Primary Unit of a Secondary Unit of a Multiple Selection n P Multiple Selection 44 Offline Sorter Offline Spike Sorting Software Waveforms View 3 5 Waveforms View The Waveforms view shows the superimposed waveforms aligned by default at the threshold crossing times Offline Sorter Software draws the waveforms assigned to units in the colors associated with those units Offline Sorter Software also draws the template for each unit in a contrasting color Waveforms 10x F x gS i Add Unit Delete Unit Add Wfs Remove Wfs Invalidate Wfs amp ee ee el et oo oe Sins aces set een chee me o O Ef CR E E E AAT E Balers E A A A oy Note that by default the Waveforms view shows its toolbar and the toolbar has the Text Below Icons option checked See Section A 2 2 Toolbars Many of the common actions described below can be initiated by pressing a button in this toolbar Waveforms can be aligned at their minimum or maximum values by using the Tools Align Waveforms or the Waveforms Align menu commands See Section 4 4 4 Waveform Alignment on page 138 Among the operations commonly done from the Waveforms view are e Selecting Waveforms Left clicking in the Waveforms view selects the nearest waveform Versi
344. rtain time intervals the waveforms collected from those intervals can be invalidated simply to get them out of the way Offline Sorter Software contains a tool that enables selection of waveforms within some interval in time around an Event or between pairs of Events in the data file For more details see Section 5 8 6 Tools Interval Selection on page 264 Note Waveforms can be marked as invalid in either waveform space or in feature space CAUTION Invalidated waveforms not marked as such in output files Invalidated waveforms may not be marked as such in the output file for some file formats notably PLX and PL2 When the file is read in again the invalidated waveforms will be valid and unsorted again However the File Export to New PLX and File Export to New PL2 features offer an option to not write invalidated waveforms to the output file 130 Offline Sorter Offline Spike Sorting Software Preparation For Sorting 4 4 2 1 Marking Artifact Waveforms as Invalid using Waveforms View In the Waveforms view shown below two artifacts arrows can be clearly identified due to their sharp waveform shapes 2500 my 1000 usec To mark the artifact waveforms as invalid and remove them from the display y lInvalidate Wfs E Waveforms 1 Click Invalidate Wfs 2 Move the pointer to the Waveforms View lt The pointer changes to a drawing hand 3 Click the left mous
345. rted Spike Timestamps Export Waveforms 7 Export Continuous Data Channels v Export Up Sampled Filtered Continuous Data Export Only the Selected Continuous Sources Id Source Chs Freq gt 101 SPK 1 64 40000 x 102 FP 65 128 1000 Export Unit Templates as Single Waveform Variables 7 Export Time Segments as Interval Variables Time Range to Export Entire File Only Data from within the Time Range From sec 0 To sec 6 034225 D Only Data from within the Time Segment i Cancel Time Range Limit Also Affects Continuous Events 2 In the What to Export area click the additional items to export Spike timestamps for sorted units are always exported to the NeuroExplorer NEX file You can add any of the following items Export Unsorted Spike Timestamps Writes the timestamps for the unsorted waveforms as NeuroExplorer variables The NeuroExplorer variables for unsorted timestamps have a U appended to denote unsorted waveforms Export Waveforms Exports the waveforms for each unit as a new set of NeuroExplorer waveform variables that are named as the timestamp variable name with _wf appended Export Continuous Data Channels Exports any continuous data channels to the NeuroExplorer file as NEX continuous variables Export Up Sampled Filtered Continuous Data Select this item to store the up sampled if relevant and low cut filtered if relevant version of th
346. rter Software contains a mechanism to mark waveforms as being invalid so that they are not considered in subsequent operations such as spike sorting There are several reasons to do this 1 Waveforms with shapes uncharacteristic of neuronal action potentials such as those caused by stimulus or movement artifacts can be marked as invalid and removed before spike sorting is performed You can remove these waveforms by following the procedure in the Section 4 4 2 1 Marking Artifact Version 4 0 129 4 Spike Sorting Step by Step AN Waveforms as Invalid using Waveforms View on page 131 In general artifact waveforms are best invalidated because they can significantly influence the principal components and the display ranges for the other views invalidated waveforms are not used when auto scaling and auto zooming operations are performed by Offline Sorter Software 2 Waveforms can be just noise that satisfied the threshold but that did not represent action potentials The easiest way to invalidate these noise waveforms is to use the Clusters views as outlined in the Section 4 4 2 2 Marking Noise Waveforms as Invalid Using Clusters Views on page 132 This step is optional you can also just leave these noise waveforms as unsorted 3 Another reason to mark waveforms as invalid is that they are simply not of interest For example if the experimental animal was not exhibiting interesting behavior during ce
347. s Analysis Operation 101 Rescuing Overlaps 104 Chapter 4 Spike Sorting Step by Step 107 Procedure Summary 109 Waveform Acquisition From Continuously Recorded Data 110 Opening the Digitized Data Files 110 Selecting the Continuous Data Channel 111 Specifying Up Sampling 114 Adjusting Gains 115 Selecting a Digital Filter Low cut or High cut 118 Specifying the Waveform Detection Options 120 Extracting the Thresholded Waveforms 123 Waveform Acquisition From Spike Waveform Data 124 Opening the Spike Waveform Data File 124 Stereotrode and Tetrode Data Type Selection 125 Selecting the Data Channel 125 Preparation For Sorting 127 Data Inspection 127 Waveforms View 127 Clusters Views 128 Timeline View 129 Invalidating Waveforms 129 Marking Artifact Waveforms as Invalid using Waveforms View 131 Marking Noise Waveforms as Invalid Using Clusters Views 132 Invalidating All Except 135 Automatically Invalidating Artifacts 135 Examining Events 137 Waveform Alignment 138 Adjusting the Sort Range 142 Selecting Features to Use For Sorting 143 Spike Sorting 143 Manual Semi Automatic and Automatic Clustering Methods 143 Selecting the Sorting Method 146 Manual Clustering using the Contours Method 146 Adding a Unit 148 Adjusting the Sorting 149 Manual Clustering using the Waveform Crossing Method 153 Adding a Unit 153 Adjusting the Sorting 154 Version 4 0 xi Semi Automatic Clustering using Templates Managing Unit Templates and Fit Tolerances Re
348. s Help Value Hardware Resources OS Name Microsoft Windows XP Professional Components Version 5 1 2600 Service Pack 2 Build 2600 Software Environment OS Manufacturer Microsoft Corporation Intemet Settings System Name JIMP 5 _ System Manufacturer Dell Computer Corporation iE on eal System Model Precision WorkStation 340 System Type X86 based PC j Processor x86 Family 15 Model 2 Stepping 4 Genuineln BIOS Version Date Dell Computer Corporation A03 4 18 2002 SMBIOS Version 23 Windows Directory CAWINDOWS System Directory C WINDOWS system32 Boot Device Device Harddisk Volume 1 Locale United States Hardware Abstraction Layer Version 5 1 2600 2180 amp xpsp_sp2_rtm 04C User Name plexoninc Jim Time Zone Central Standard Time Total Physical Maman 1726 00 MR w lt gt a C Search selected category only _ Search category names only A 3 4 Messages Window The Messages window displays a log of timestamps and associated application events that Plexon Support can use for troubleshooting purposes Note The Messages window is not available for all applications Time Message 09 54 2 06 CinePlex Markup version 1 2 0 BETA Starting 09 54 2 07 Using C Program Files Plexon Inc CinePlex as main directory 09 54 16 37 Reading neural data file C Program Files Plexon Inc CinePlex Temp CineplexQuickSt 09 54 16 40 Reading video data file C Program Files Plexon Inc CinePlex Temp Cin
349. s In the Clusters Views on page 151 4 5 12 Parameter Scans Offline Sorter Software contains a tool to explore different sorting scenarios by scanning the values for parameters that affect the sorting over a range of values re running the sorting and recording the Sorting Quality Statistics at each step Select the Scan option in the Info Grid Views menu to display the Scan view Sorting Parameter Scan for Channel1 Scan Parzen Radius Multiplier for Valley Seeking i l iw Use 3D 0 500000 End 2 500000 Step By 0 500000 i V Normalize Start Scan Pseudo F DB Dunn p i Sorted LRatio IsoDist 3 6854 0 472793 0 993611 34 9613 1824237 7 15 5225 13 61679 0 419862 1 05081 41 6739 92 5276 6 123 2003 3 60846 0 42844 0 997185 38 6433 93 8921 0 22 918 3 55551 0 39106 1 12614 4 22614 94 7368 0 36 0794 3 50044 0 418005 1 08234 1 46185 195 744 0 52 5887 Bestby J3 0 15 _ Re sort Using This Value Best by Pseudo F 0 H il Re sort Using This Value Best by D B i __Re sort Using This Value Best by Dunn i i Re sort Using This Value Best by p i i il Re sort Using This Value Best by LRatio 3 Re sort Using This Value Best by Iso Dist ___Re sort Using This Value You can do several different types of scans which can be selected from the Type droplist For each type click Start Scan to cause the procedure i
350. s do The search for seed points starts at the top of the list sorted according to the number of neighbors so the seed points are points near the centers of the densest clusters Points with less than a certain minimum number of neighbors cannot become seed points Then initial clusters are formed from each seed points and all of the seed point s neighbors Also if any of the seed point s neighbors are themselves seed points their initial clusters are combined At this stage the clusters consist of a circle or sphere in 3D of points centered around each density maximum with a radius of roughly the critical neighbor distance with the other points unassigned The unassigned points are then assigned to clusters using a modified Fukanaga algorithm This is an iterative algorithm at each step unassigned points are assigned to the cluster that the majority of its neighbors belong to This continues until points stop being assigned or a maximum number of iterations is completed References Fukunaga Keinosuke Introduction to Statistical Pattern Recognition Chapter 11 Academic Press 1972 6 5 8 T Distribution E M Algorithm A variant of the E M algorithm by Shoham et al see references that follow This algorithm has two important features 1 Instead of fitting a mixture of normal Gaussian distributions T Distributions are used instead The T Distribution is similar to a Gaussian except that the tails are fatter This c
351. s from one another For low amplitude noise the noise cluster is centered close to the origin 0 0 in principal component space If the noise cluster is localized near zero and isolated it can be removed from the principal component calculations so that the new principal components maximally separate the remaining unit clusters from one another You can then select the maximally separated unit clusters by using the Contour method To invalidate the noise waveforms near zero 1 Click Invalidate Wfs Y Z X Add Unit Find Units Dels Po i i amp y Invalidate Wfs E 132 Offline Sorter Offline Spike Sorting Software Preparation For Sorting 2 Move the mouse pointer to the Clusters View lt The pointer changes to a drawing hand If the standard deviation method is used the standard deviation may be calculated via the MAD technique see Section 6 5 3 MAD Calculation on page 301 3 Click the left mouse button and drag a line around the invalid noise waveforms near zero In the following 2D Clusters view the noise cluster near the origin is invalidated gray Add Unit PC2 Note You can also use the 3D Clusters view for the following procedure To remove the invalid waveforms from the display Set the Invalid w f parameter in the Control Grid to Hide pe ee My See Draw Grid Draw Thr aE ae 3 reao re E In Version 4 0 133 4
352. s in the channel does not change That is changing the channel or re extracting waveforms with a different threshold thus resulting in a different number of spikes will invalidate any previously saved SORT files 5 6 8 Sort Save Sort Info to TPL File and Sort Sort Using TPL Flle Select these menu items to save sorting information to a TPL file or to apply an existing TPL file to the current channel See Section 4 8 4 Saving Template Files on page 212 for details 5 6 9 Sort Sort Using NEX File Offline Sorter Software can also resort a file by using the unit assignments that are saved in a NEX file This function is only useful if applied to the data file originally used to produce the NEX file You can use this feature to facilitate retaining only the original raw data file on disk rather than retaining the raw data file plus possibly several versions of the data file sorted slightly differently To apply the sorting from a NEX file to the currently loaded file select Sort Sort Using Saved NEX File The following dialog appears Version 4 0 255 5 Menu Options Sort From NEX File This operation will read an existing NEX file and re apply the sorting to the currently loaded data This operation only makes sense when the NEX file was produced by Offline Sorter from the same data file WARNING Proceeding will delete any existing sorting done on this file and the operation cannot be undone
353. s is controlled by the Initial Fit Tolerances for Band Units section of the Tools Options Sort dialog The band is set either to a specified number of standard deviations calculated at each sample position using the crossed waveforms or is set such that every crossed waveform is within the initial band Version 4 0 169 4 Spike Sorting Step by Step i amp x 2 i Add Unit Delete Unit Add Wfs Remove Wfs Invalidate Wfs i Tolerances for Unita Adjust the template and band heights Each small box on the screen can be grabbed and dragged with the mouse to reshape both the unit template and the heights of the band at each sample location in the waveform Note that the band is always symmetric around the template dragging the top of band position will simultaneously adjust the bottom of band position Alternately the band heights can be adjusted systematically using the Tolerances for Unit up down arrows on the secondary toolbar underneath the main toolbar When using these up down arrows the general shape of the band is preserved but all points in the band are moved slightly closer to down arrow or away from up arrow the unit template 170 Offline Sorter Offline Spike Sorting Software Spike Sorting k x Add Unit Delete Unit Add Wfs Remove Wfs Invalidate Wfs iTolerances for Unita Note The Bands sorting algorithm is greedy in that a waveform is sorted into the
354. s than the number of waveforms In general if the waveform count on a channel exceeds the Max w f draw setting do not select the Use only currently visible waveforms checkbox in the Tools Options General tab that selection can result in valid waveforms being unassigned when selecting units by using the Waveform Crossing or Contour sorting methods Sort Start and Sort End are the lower bound and upper bound of the time interval See Adjusting the Sort Range on page 142 of the waveform that Offline Sorter Software uses to calculate principal components and feature values and in Section 6 5 Details of the Sorting Algorithms on page 299 The default value for Sort Start is zero The default value for the Sort End is the total waveform length Offline Sorter Software only uses the time points of the waveforms between Sort Start and Sort End in the Template Sorting algorithm in the recalculation of the principal component scores and in the calculation of feature vectors For options in principal component calculations see Section 4 4 5 Adjusting the Sort Range on page 142 Slice 1 Slice 2 Slice 3 Slice 4 are the time points in the waveform slices in time that Offline Sorter Software uses to calculate the Slice feature A vertical line in the Waveforms view represents the Slice time It is possible to view the voltage of the waveforms at the Slice time point on any axis of the Clusters views and it can be used in the
355. s the color used to draw them Clicking on a Display Color cell allows changing the color Note also that the default Display Color used for all newly created time segments can be specified in the Tools Options Colors tab of the Options Dialog The Waveforms column shows the total number of valid waveforms within the time segment and the narrow column next to it shows the number of Invalid waveforms in the time segment The Unsorted and Unit columns show the count of unsorted waveforms and the number of waveforms sorted into each unit within the time segment The rest of the columns show the sort quality parameters for the waveforms in the time segment The following functions are available after right clicking on a time segment e Make Selected Segment Current Makes the highlighted segment into the current time segment which is denoted with a gt gt in the first column e Delete Selected Segments Can delete one or more highlighted Time Segments The Entire File time segment is special and cannot be deleted e Create New Segments Shows the Create New time segments dialog see Section 5 5 4 Waveforms Create Time Segments on page 243 e Show 3D Per Segment Statistics Controls whether the sort quality parameters are computed using the 2D or 3D feature space e Show Normalized Per Segment Statistics Controls whether the raw or normalized version of the sort quality parameters are displayed see discussion
356. s view Add Unit Delete Selected Unit EA Info BA 2D Clusters HH Features E 3D Clusters Show Tetrode Electrode Combinations shows the 6 permutations of 4 tetrode electrodes using a tetrode per electrode feature value Per Electrode Peak Height by default For example this mode shows plots of Peak Height for Electrode 1 vs Peak Height for Electrode 2 Peak Height for Electrode 1 vs Peak Height for Electrode 3 and Peak Height for Electrode 3 vs Peak Height for Electrode 4 This is the default mode for Tetrode Data Types In 82 Offline Sorter Offline Spike Sorting Software Feature vs Feature View this mode press the N or P keys to go to the next or previous channel as in the 2D Clusters view Feature vs Feature Valley EL 2 Valley EL 3 m hog TT TT gt gt 2 2 z z Valley EL 2 Valley EL 4 Valley EL 4 Valley EL 1 Valley EL 3 To select which Per Electrode feature is used for the Tetrode electrode display mode select that feature for the X Axis in the 2D Clusters view using the feature droplists in the toolbar For example if one selects PC 1 EL 1 for the X Axis of the 2D Clusters view the Feature vs Feature view operating in Tetrode electrode display mode shows the plots PC1 EL 1 vs PC1 EL 2 PC1 EL 1 vs PC1 El 3 and PC1 EL 3 vs PC1 EL 4 If one selects a non Per Electrode feature for the X Axis in the 2D Clusters view the tetrode display uses Per Electrode Peak Hei
357. separate waveforms each with a different timestamp that is determined by the offsets associated with the Overlap Template that the overlapping waveform matched The set of samples for each of the rescued waveforms are extracted from the continuous data by looking at the continuous data at positions determined by the offsets Note that this includes samples that are outside of the Waveform Window of the original overlapping spike 104 Offline Sorter Offline Spike Sorting Software Overlaps Analysis View Then for each rescued overlapping waveform Offline Sorter Software attempts to remove the effects of the unit superposition by using the unit templates Using the time shifts associated with the matching Overlap Template Offline Sorter Software can remove the contamination of the first waveform by subtracting off a time shifted version of the second unit template And similarly it can remove the effect of the first waveform impinging on the second rescued waveform by subtracting off the first unit template Because the Overlaps Analysis procedure rests on a number of assumptions best results will be obtained if a specific workflow is followed This workflow is outlined in Section 4 6 5 Rescuing Overlapping Waveforms on page 194 Version 4 0 105 3 Views and Displays 106 Offline Sorter Offline Spike Sorting Software Plexon Inc Chapter 4 Spike Sorting Step by Step di Procedir SUMMAN sa
358. sholding 77 97 310 time segments 243 Time Segments view 34 Timeline view 48 129 timestamps 205 timestamps exporting 231 toolbars 17 Tools menu 262 U unit display 42 Index 3 Units menu 257 Units view 42 units cleaning 184 units sorting 142 updates software 5 up sampling 114 user interface see also Appendix A 17 V Valley Seeking algorithm 304 version of software 274 View menu 21 237 views 18 21 W waveform alignment 138 waveform detection 120 Waveform Detection view 74 waveform detection automatic 249 waveform display 184 Waveform Inspection view 73 waveform selection options 186 Waveforms menu 241 Waveforms view 23 45 127 waveforms extracting 123 waveforms invalidating 129 waveforms RMSE summed RMS 86 waveforms selection 199 Windows menu 272 Index 4 Offline Sorter Offline Spike Sorting Software Offline Sorter Offline Spike Sorting Software User Guide Plexon Inc 6500 Greenville Avenue Suite 700 Dallas Texas 75206 United States of America Telephone 1 214 369 4957 Fax 1 214 369 1775 www plexon com Copyright 1983 2015 Plexon Inc All Rights Reserved Plexon Inc Proprietary Document Number OFSMN0004a Document Status Standard Software Version 4 0 Date April 2015 ALEeXONn
359. signal exceeds either threshold a logical OR of thresholds Note Always set Thr 2 Thr This checkbox always sets the second threshold to be the negative value of the first threshold making the dual thresholds symmetric around zero Using dual thresholds is not recommended except for unusual cases A detailed understanding of waveform alignment issues is required to avoid being fooled as dual thresholds can easily make a single unit appear as two distinct units depending on which part of the waveform crosses threshold first e Continuously Sample Raw Signal this option is generally useful in special situations only It will continuously trigger spikes so that every continuous sample is included into exactly one spike Alignment During Detection Raw Signal Only to enable spike alignment during detection click the Enable checkbox When active spikes will be aligned to the largest peak voltage of each spike instead of to the sample where the spike crossed threshold Alignment during detection is generally much preferable to aligning spikes after detection because the Alignment During Detection algorithm can sample the full waveform from the continuous data sample instead of having to shift in zeroes at the edges The algorithm can take any minimum or maximum voltage value as the alignment point the Either Side radio button or can insist that the minimum or maximum voltage is on the same side of zero as is the thresh
360. sion 4 0 Appendix A Navigating the Plexon UI Clicking the Customize menu command will open the Customize dialog box This dialog box is common to Plexon software applications but may have different content on the tabs according to the software application Customize Toolbars Commands Keyboard Run Menu Options Toolbars en ea Standard Edit _ amp nimate Looping Find Pool Views For details on using the Customize dialog box see Section A 4 Customization on page 18 A 2 3 Windows The illustration below shows a typical window title bar It contains from left to right a Title a Auto Hide button a Maximize button and a Close button i Position 1 Marker Occu O Time x Y a g Auto Hide Button The Auto Hide button pins a window to the screen to keep it visible or rolls up a visible window into a tab When the window is pinned the Auto Hide button points in a vertical direction If the window is rolled up the Auto Hide button points in a horizontal direction o Maximize Button The Maximize button may not appear on all windows It is the standard Windows maximize button Clicking the Maximize button on a window will maximize the original window and hide other windows occupying the same horizontal or vertical space Clicking the Maximize button again will restore the previous layout When clicked the image on the button toggles between one window and ove
361. sort quality metrics 309 PL2 file exporting to 232 PL2 files opening 226 PLX file exporting to 231 PowerPoint send sort summaries 236 principal components 297 principal components analysis PCA 312 printing 236 Pseudo F statistic 308 Q quality of sorting J3 measure 308 Quick Batch Reference 274 quick key reference and Help files 273 R Rasters view 92 raw channels 277 RMSE summed root mean squared 86 rotate the display 66 Run menu 272 S saving and exporting results 14 Scan view 36 179 screen elements 17 Select menu 240 semi automatic clustering 157 software installation 2 software updates 5 software version 274 Sort 251 Sort Details view 35 Sort menu 251 sort method 38 Version 4 0 sort quality 97 sort partial 306 sorting algorithms 10 299 sorting features 294 sorting quality statistics 306 sorting spikes 143 sorting statistics 200 Sorting Summary view 84 Sorting Summary customizing 87 sorting techniques 10 sorting units 142 Sources view 27 spike sorting 109 143 algorithms 10 outputs 14 spikes stereotrode and tetrode 291 statistics feedback 203 statistics sorting 200 Stats view 36 stereotrodes and tetrodes 78 125 291 292 support Plexon technical support 5 Surface view 69 system overview 8 9 T technical support 5 Template sorting algorithm 300 templates 157 Templates view 37 terminology 16 tetrodes see stereotrodes and tetrodes Threshold Scan Graph view 97 thre
362. sorting algorithms Outlier Thr Outlier Threshold can be used for several purposes The value in this field is in units of standard deviations sigmas When Offline Sorter Software renders ellipses in the Clusters views this field controls where Offline Sorter Software draws the lines or surfaces For the Semi Automatic Clustering using K Means sorting algorithm and for the Tools Remove Outliers menu selection this field controls the removal of outliers The K Means clustering algorithm assigns every waveform to a cluster To eliminate the outliers Offline Sorter Software calculates the standard deviation o of the distance from all points in the cluster to its center for each cluster Then if the distance from a given point to the cluster center is more than o Outlier_Threshold Offline Sorter Software removes the point from the cluster Parzen Mult D O F Mult or Beta Only one of these values appears which depends upon the sorting method currently selected All the values control parameters in the sorting algorithms as follows The Automatic Clustering using Valley Seeking Method algorithm works by counting neighbors that are within the Parzen radius times this Parzen Mult multiplier around the point in feature space If the value is decreased it generally results in more numerous smaller 40 Offline Sorter Offline Spike Sorting Software Control Grid clusters The Automatic Clustering using T Distribution E
363. starting with Unit a for which it passes through all lines If a waveform passes through all of the lines for several units it is assigned the lowest lettered unit 4 5 7 Manual Clustering using the Bands Method Select the Bands method of sorting by selecting Bands in the Sort Method droplist in the Control Grid or by selecting the Sort Change Sort Method Use Bands menu item The Bands algorithm works in waveform space not in feature space The goal of the algorithm is to define a template waveform shape for each unit along with an adjustable band or corridor around the template through which all waveforms that are sorted into that unit must be contained To add a unit using the Bands sorting method perform the following steps 168 Offline Sorter Offline Spike Sorting Software Spike Sorting 1 Press the Add Unit button in the Waveforms view invalidate Wis E or right click in the Waveforms view and select the Add Unit menu command The cursor turns into a drawing hand 2 Define the unit template waveform by crossing a bundle of waveforms exactly as described in Section 4 5 3 Manual Clustering using the Waveform Crossing Method on page 153 As soon as the mouse button is released after a bundle of waveforms is crossed a new unit template waveform is defined from the crossed waveforms and an initial band is formed around the template How the initial band is formed from the crossed waveform
364. t Apply Template Sorting with Adaptive Templates or by clicking the a button on the toolbar After Adaptive Template Sorting has been applied to a channel there are two views available in Offline Sorter Software that are designed to show how the templates for each unit evolved as a function of time Adaptive Templates History in the Info Grid Views shows each template that was used during the adaptive template sort on each unit along with the time at which each template was put into effect Version 4 0 161 4 Spike Sorting Step by Step PAOR ae EE S E Adag emplate liste Adaptive Templates for Channel 4 in A D counts To display the Adaptive Templates History view select View Info Grid Views Adaptive Templates History from the main menu The grid is divided into sections on section for each unit Within each section each row corresponds to a new template that was put into effect during the Adaptive Template Sorting On each row the first entry is the time in seconds at which the template was put into effect The other N columns show each sample value for the template waveform where N is the number of samples per waveform By default the sample values are shown in raw a d counts but they can be displayed in microvolts by selecting Show Templates in Microvolts from the right click context menu The Templates vs Time view shows a graphical depiction of the same information that is displayed in table form in t
365. t File Import menu command and select the vendor Once the data file is opened the waveforms are viewable and ready for sorting 4 3 2 Stereotrode and Tetrode Data Type Selection Offline Sorter Software can sort files with signals from single electrodes stereotrodes or tetrodes see also Section 6 3 Stereotrode and Tetrode Data on page 291 The Data Type Single Stereotrode Tetrode is displayed in the Control Grid Zoom 1 0 Max wif draw 100000 For Plexon PLX and PL2 files DataWave UFF files and Neuralynx files Offline Sorter Software automatically detects stereotrode or tetrode data when the file is opened and sets the Data Type accordingly For all other file types Offline Sorter Software initially assumes single channel data If the data type displayed in the Control Grid is not appropriate for the data file opened Offline Sorter Software can sometimes convert the data type For a description of this procedure see Section 6 2 2 Converting Data Type on page 288 4 3 3 Selecting the Data Channel Offline Sorter Software can be configured to automatically load the first data channel into memory immediately by checking the Automatically Load First Channel after Opening a File checkbox in the General tab of the Tools Data Import Options dialog Version 4 0 125 4 Spike Sorting Step by Step To select a particular data channel 1 Click on the Channel drop list box in the Control Grid shows
366. t except those that occur within the specified time span Occur between a time offset from Event A and a time offset from Event B This selection enables both the Event A and Event B droplists as Version 4 0 265 5 Menu Options well as the offsets This mode is like the first option except the interval between the start and stop events extends by a time offset The waveforms are kept if they occur between the time of Event A plus the first offset and the time of Event B plus the second offset e Occur within an arbitrary range of timestamps With this selection no events need to be selected only the beginning and end of a time interval as measured from the beginning of the file timestamp 0 need to be specified All waveforms that occur outside of this time range are invalidated e Occur outside of an arbitrary range of timestamps With this selection no events need to be selected in this case only the beginning and end of a time interval as measured from the beginning of the file timestamp 0 need to be specified All waveforms that occur within this time range are invalidated This is the inverse of the above option e Occur outside of time ranges read in from a file This item selects and reads a text file that contains ranges of times to invalidate Each line contains a channel number followed by the start and end times for the invalidation range which are separated by a space tab or comma The times a
367. t the Standard E M algorithm is run to sort the spikes e Scan Random Seed for T Dist E M The first step of the T Dist E M fully automated sorting method is actually a K Means sort using the number of cluster centers specified in Tools Options Sort Number of seed clusters for T Distribution algorithm using the Seed cluster pattern specified on the same Options page This scan replaces this initial seed cluster placement with a random seed cluster placement again using the random number seed that is the current scan value This method also allows specifying a fixed D O F Multiplier to be used for the T Distribution E M sort e Scan Beta for Std E M This scan runs the Beta parameter for the Std E M algorithm of the scanning range A fixed value for the number of clusters can be specified 4 5 13 Automatic Clustering Using the Scanning Methods Offline Sorter Software also offers a family of automatic sorting methods that are based on scanning sort parameters At each value for the sort parameter one of the other sorting methods is run and the sort quality metrics are evaluated The sorting that produced the best value of a specified sort quality metric is then chosen as the sorting for that channel The options that control the behavior of the scanning sorting methods are set in the Tools Options Sort tab 182 Offline Sorter Offline Spike Sorting Software Spike Sorting Seed cluster pattem Circular D Lin
368. ta Type is changed the channel map will reconfigure to show the channel mapping for the Source with the new Data Type and an Apply button appears This is a form of preview for the new channel mapping the re mapping does not take place until the Apply button is pressed When a different Data Type is selected and the Apply button is pressed Offline Sorter Software performs a conversion algorithm on the data This conversion causes all the data to be reloaded and all sorted unit assignments and parameters to be lost CAUTION Pressing the Apply button reloads data and resets parameters Use the Apply button with caution Verify that you want to reload data and drop the current sorted unit assignments and parameter values The algorithm used to convert data type is as follows using the conversion of single channel data into tetrode data as an example 1 Group the channels into groups of four two for stereotrode data If the original single channel data file has N channels there will be N 4 tetrode channels N 2 for stereotrode after the conversion Version 4 0 289 6 Reference Topics 2 Using the first channel in each group step through each spike in the channel and obtain its timestamp Find the corresponding spikes with the same time stamp on the other three channels in the group Failure to find a spike on the other channels with the same timestamp results in a dropped waveform A count of dropped waveforms app
369. ta points It is so named because for each iteration the algorithm goes through two phases an Expectation calculation followed by a Maximization Version 4 0 303 6 Reference Topics phase You can find the derivation of the equations for the algorithm in many sources some of which are listed below The standard E M algorithm requires the selection of the number of Gaussians to be fit i e the number of units and an initial starting assignment of points to clusters In Offline Sorter Software if a Continue Sort is being performed see Section 5 6 2 Sort Perform Automatic Sorting on page 251 the existing point assignments and number of clusters is used If a normal Standard E M sort is requested the user is required to specify starting cluster centers and a K Means sort is used to get the initial assignment of points to clusters Offline Sorter Software actually implements a variant of the Standard E M algorithm called REM 2 as discussed in the Sahani reference below With this variant a new parameter beta is introduced that influences the convergence of the E M algorithm In the Sahani reference this beta parameter is systematically decreased as the algorithm progresses in an annealing schedule which causes clusters to be frozen out as phase transitions are encountered at certain values of beta When the beta value is set to 1 0 the REM 2 method becomes equivalent to the Standard E M algorithm
370. tails see Section 4 4 4 Waveform Alignment on page 138 5 8 3 Tools Adjust Gains This menu item shows the Adjust Gains dialog box as described in Section 4 2 4 Adjusting Gains on page 115 5 8 4 Tools Invert Waveforms This menu item is for special use cases only It flips the polarity of all waveform data 5 8 5 Tools Re Threshold Waveforms In addition to the ability of Offline Sorter Software to threshold continuously recorded data Offline Sorter Software can also simulate the effect of setting a different voltage threshold for the detection of spike waveforms All spike waveforms that do not cross the new threshold are marked as invalid Obviously setting a threshold lower than what was in effect when the file was created does not put more waveforms into the file Version 4 0 263 5 Menu Options The first step in running the re thresholding tool is to make the threshold marker line a dashed white horizontal line appear on the Waveforms view There are two ways to accomplish this e Check the Draw Thr checkbox on the control panel This permanently enables the display of the threshold marker line on the Waveform display e Select the Tools Re Threshold Waveforms menu item This temporarily shows the threshold marker line The line disappears after the re thresholding operation completes Outlier Thr Parzen Mut View wit Cluster View Contin View Draw Grid Draw Thr Refr ISI us 10
371. takes place You can undo the operation by using the Undo mechanism or by selecting the Waveforms Mark All Waveforms Valid menu selection 266 Offline Sorter Offline Spike Sorting Software Tools Menu 5 8 7 Tools Remove Short ISI Waveforms Invokes the outlier removal algorithm described in Section 4 6 4 1 Automatic Short ISI Spike Removal on page 191 5 8 8 Tools Remove Outliers Invokes the outlier removal algorithm described in Section 4 6 3 1 Automatic Outlier Removal Tool on page 187 5 8 9 Tools Assign Unsorted to Closest Unit After some operations e g Valley Seeking Sorting it is desirable to assign unsorted waveforms membership in the nearest unit subject to the Outlier Threshold Select Tools Assign Unsorted To Closest Unit to perform this operation The algorithm this tool uses is as follows e Calculate the centroid and standard deviations along each principal axis for each existing unit in the appropriate 2D or 3D feature space If the 3D Clusters view was showing when the tool was selected then 3D feature space is used otherwise 2D feature space is used e Calculate the Mahalanobis distance from each unassigned waveform point to all the unit centroids in order to find the closest unit Note The units of Mahalanobis distance is standard deviations e If the distance to the closest unit is less than the Outlier Threshold for that unit assign the waveform to that unit Note
372. tandard E M on page 175 Selecting this menu item will clear the unit centers to allow the user to start again e Delete Selected Unit removes the unit currently highlighted in the Units Display This is the same as pressing the Delete Unit button in the Per View Toolbar e Delete All Units removes all units currently defined for the channel Note The above two options are also available from the Units menu on the main menu bar e Add Waveforms to the Selected Unit allows adding new waveforms to an existing unit by circling them This is the same as pressing the Add Wfs button in the Per View Toolbar 60 Offline Sorter Offline Spike Sorting Software 2D and 3D Clusters Views e Remove Waveforms from the Selected Unit allows removing waveforms from an existing unit by circling them This is the same as pressing the Remove Wfs button in the Per View Toolbar e Invalidate Waveforms allows marking waveforms as being invalid by circling them This is the same as pressing the Invalidate Wfs button in the Per View Toolbar Show Buttons shows the buttons as described in Section 3 7 2 6 Per View Toolbars Hints and Statistics Display on page 58 e Show Hints shows text hints as described in Section 3 7 2 6 Per View Toolbars Hints and Statistics Display on page 58 e Show Statistics shows the Sort Quality Statistics in the Hints area When Hints are being shown they use the same area
373. tandard deviation of waveforms radio button the Fit Tolerance will be calculated by finding the standard deviation of the difference between each waveform in the unit and the unit template and setting the Fit Tolerance to a constant multiple of that value When using this option a refinement is available to how the standard deviation is calculated By checking the Use MAD checkbox the Median Absolute Deviation MAD technique is used to calculate a modified standard deviation that is less sensitive to outliers See Section 6 5 3 MAD Calculation on page 301 for details Note Even immediately after the unit templates and the Fit Tolerances are calculated from the existing units performing a Template sort may not accurately reproduce the current sorting 4 5 4 2 Re applying Template Sorting Regardless of how clusters are created you can perform Template Sorting on the clusters using the current unit templates and Fit Tolerances To perform a template sort select Sort Apply Template Sorting with Current Templates or click the Run Template Sort button on the toolbar For a discussion of how Offline Sorter Software creates and maintains unit templates and Fit Tolerances see Section 4 5 4 1 Managing Unit Templates and Fit Tolerances on page 158 4 5 4 3 Applying Adaptive Template Sorting Similarly Offline Sorter Software can apply a variant of Template Sorting to the existing clusters where the template is modified as the T
374. tation options Note These options also control how strobed events are exported into NeuroExplorer files For Plexon PL2 files there is a concept of an event Source Event Sources are similar to continuous or spike Sources they are groups of event Channels where each channel is a separate Event The Sources view will list the Events Sources when a PL2 file is opened a EE E E E EE SPK Spike H 40000 7 Cont 1000 8 Event i3 3 4 Single bitevents Event 10 5 Other events Event i43 an 6 CinePlex Data Event 12 There are convenient context menu options available in the Sources view when an event Source is selected Enable All Events from this Source and Disable All Events from this Source are equivalent to checking or unchecking all the Show column checkboxes in the Events view for all Events belonging to the selected event Source Selecting Enable Only Events from this Source is equivalent to checking all the Show column checkboxes for Events in the selected event Source and unchecking all the checkboxes for the Events from other event Sources 4 4 4 Waveform Alignment After detection and extraction the waveforms are initially aligned at the point of threshold crossing in the Waveforms view below left The principal components are calculated using the threshold aligned waveforms You can select 138 Offline Sorter Offline Spike Sorting Software Preparation For Sorting the projections onto
375. te For New Unit And Sort from the right click shortcut menu 4 5 4 1 Managing Unit Templates and Fit Tolerances Offline Sorter Software allows control over how and when unit templates are updated and used Besides being displayed in the Units view the unit templates also appear numerically in the Templates tab of Info Grid Views By default Offline Sorter Software always recalculates the unit template for a unit after any change to the sorting is made Offline Sorter Software calculates the unit template for a unit by averaging together all of the waveforms in that unit It will also recalculate the unit templates when a file is first loaded However this can be slightly confusing when loading a PLX or PL2 file that was collected by the Plexon MAP system or OmniPlex System using Template Sorting as the recalculated unit templates in Offline Sorter Software may be slightly different than the unit templates used by the MAP system or OmniPlex System for online sorting You can disable the automatic recalculation of unit templates by clicking to clear the Units Automatically Recalculate Unit Templates after Each Change menu item or by clicking to clear the Automatically Recalculate Unit Templates checkbox on the Templates tab of the Tools Options dialog box In this case the unit templates are only recalculated when the user explicitly requests it by selecting Units Calculate Unit Templates from Existing Units or clicks the Recalculate
376. te Sort it determines which waveforms are part of the units and so contribute to the unit tem plates and which waveforms are left unsorted and thus considered as potential overlapping waveforms Second it determines how readily a waveform will be considered matched to an Overlap Template to be rescued an overlapping waveform must match an Overlap Template to within the tolerance For convenience there are controls on the Overlaps Analysis view that allow quickly adjusting the Tolerance for all units simultaneously and resorting the units by pressing the Template Sort Units button Note that there is also an implicit assumption that the same Tolerance has been applied to all units as is enforced if the above controls are used Run the Overlaps Analysis First decide on a Minimum Overlap value Small values are not recommended otherwise first few and last few Overlap Templates will likely match the original template units Remember that the Minimum Overlap is specified in samples The total number of samples in a waveform can be determined by checking the Waveform Detection Options dialog where the number of samples corresponding to the Waveform Length is shown see above screenshot Pressing the Run Overlaps Analysis button will create the Overlap Templates and populate the grid Checking the Show on Clusters Views checkbox will allow inspecting how the overlaps might appear on the Clusters views Note that after pressing the Ru
377. teeeees A 11 Aa Window Menisin A 11 AGa Ran Meni Sri toads A 13 Fiche PISS OT a isa saeee ea pands Sestvae tadonraeaalonnraeaaaane A 13 A234 Messages Window cc0 s0cccctsceieeesdaeceeeesdiaceeeeeiaianes A 16 ASO FITC GK ISIS ccc casceccceccancnescnestxertenetanuieientmmmnance A 17 A 3 6 Current Selections iiscnniivevemaienenssniser naiasee aaa A 17 Pad a U iai aaa S aaa A 17 AA Cu ustomizalon ecarinascedacixasseadakisvocsdaxixandadediatareladaxtemnacacdiuaneds A 18 A 4 1 Toolbars CUStOMIZATION ssicsssisisisosirsiininiienai A 18 A 4 2 Commands Customization c cscceeeeessteeeeeeeenaees A 19 A 4 3 Keyboard Customization ccsiccscssccsvssrseceisaccensaesaaeenacs A 19 A 4 4 Run Menu Customization cccccsscceessesseeeeeeeeeaes A 21 AAS Options Customization s cccccsssarcessessnesevecsarsteteorss A 22 Version 4 0 Appendix A Navigating the Plexon UI A 1 Plexon User Interface The Plexon User Interface embodies a standard look and feel for Plexon software products To illustrate the underlying concepts behind the look and feel this appendix uses screenshots from Plexon s CinePlex Editor formerly called CinePlex Markup and CinePlex Studio formerly called CinePlex Capture applications Although there will be differences in menus and toolbars between different applications how you use handle and operate the user interface components will be fundamentally the same across future generations of Plexon
378. ter Software is to play back the data in a file in time order This is useful in determining whether waveform shapes are drifting as a function of time You can make Offline Sorter Software automatically advance time in the Timeline view by using the operations under the View Animate menu The animation functions are completely equivalent to changing the time range by using the scroll bars on the Timeline view 238 Offline Sorter Offline Spike Sorting Software View Menu za Threshold Scan Graph I Overlaps Analysis Waveforms Legacy Pog Slower Faster Reset to 1X Speed Rewind The animation operations include e Play starts animating the playback of the file but defaults advancing time at the normal rate of 1 second per second e Pause pauses the animation select it again to un pause e Stop stops the animation e Slower slows down the rate of animation by a factor of 1 2 e Faster speeds up the rate of animation by a factor of 2 e Reset to 1X Speed resets to the normal animation rate 1 second per second e Rewind resets to the beginning of the file e Reverse a toggle that makes the rate of time passage negative e g time flows backwards e Loop when checked the animation resets to the beginning of the file when the end of the file is reached To make operation much easier you can bind keystrokes to these keys by using the Keyboard tab under Windows Customize menu
379. ter inspected in Offline Sorter Software 6 9 1 5 Set The Set command changes a parameter immediately Syntax Set parameterName parameterValue e g Set ParzenRadiusMult 2 0 The available parameters are OptionsFromFile immediately sets all options to the values in the specified options file The options file can be created using the Tools All Options Save To File menu command Note that this can reset the values of any previous Set commands so it generally should come before any other Set commands This command is useful for making sure that all parameters are set to known values before executing a batch file ChanMap specifies a filename that contains a channel map to apply Having this command present will force the named channel map file to be applied to each loaded file TplFileName sets the sorting template filename to use for ForEachFile SortUsingT pl DefaultFileType specifies the file type to assume when the data file extension does not match any known extension The argument uses a numeric code from the following table to specify the file type Code File Type 0 Plexon PLX 1 reserved 2 reserved 3 Plexon PL2 4 Alpha Omega 5 CED Spike 2 6 Datawave 7 Data Translations 8 reserved 9 Multi Channel Systems 10 Panasonic MED 11 Cyberkinetics 12 Neuralynx 13 Neuroexplorer nex Version 4 0 317 6 Reference Topics Code File T
380. ters view for manipulating the eyepoint see Section 3 7 2 13 3D Clusters View Details on page 65 To show semi transparent planes at the boundaries between Time Segments select Show Time Segment Boundary Planes from the right click menu 4 5 5 Manual Clustering using the Boxes Method Select the Boxes method of sorting by selecting Boxes in the Sort Method drop list in the Control Grid or by selecting the Sort Change Sort Method Use Boxes menu item Version 4 0 163 4 Spike Sorting Step by Step The Boxes algorithm works in waveform space not in feature space The goal of the algorithm is to set two boxes in time voltage space on the Waveforms view that together define a unit To add a unit using the Boxes sorting method 1 Press the Add Unit button in the Waveforms view f amp ro 8 re Remove Wfs Invalidate Ws or right click in the Waveforms view and select the Add Unit menu command 2 Move and resize the boxes so that only the desired waveforms pass through the boxes When the cursor in inside either box it turns into a 4 sided arrow and dragging will move the box When the cursor is over one of the 8 resize handles it turns into a 2 sided arrow Then clicking and dragging will resize the box in the indicated direction 164 Offline Sorter Offline Spike Sorting Software Spike Sorting f amp amp Add Unit Delete Unit Add Wfs Remove Wfs Invalidate Ws 3 When f
381. th Adaptive Templates 254 Sort Adaptive Templates Settings 254 Sort Save Sorting to SORT File Restore Sorting from SORT File 254 Sort Save Sort Info to TPL File and Sort Sort Using TPL Flle 255 Sort Sort Using NEX File 255 Sort Delete All Companion SCAN Files 256 Sort Stop Sorting 256 Units Menu 257 Units Select Unit s 257 Units Delete Selected Unit 258 Units Delete All Units 258 Units Combine Units 258 Units Swap Units 259 Units Invalidate All Waveforms in Selected Unit 260 Units Add Waveforms to the Selected Unit 260 Units Remove Waveforms from the Selected Unit 260 Units Remove All Except Selected Waveforms from Selected Unit 260 Units Automatically Recalculate Unit Templates after Each Change 260 Units Calculate Unit Templates from Existing Units 260 Units Automatically Recalculate Fit Tolerances after Each Change 260 Units Calculate Fit Tolerances from Existing Units 261 Tools Menu 262 Tools Recalculate the Principal Components 262 Tools Align Waveforms 263 Tools Adjust Gains 263 Tools Invert Waveforms 263 Tools Re Threshold Waveforms 263 Tools Interval Selection 264 Tools Remove Short ISI Waveforms 267 Tools Remove Outliers 267 Tools Assign Unsorted to Closest Unit 267 Tools Invalidate Cross Channel Artifacts 267 Tools Invalidate High Amplitude Artifacts 267 Tools Density Leveling 268 Tools All Options 269 T
382. the 80 Offline Sorter Offline Spike Sorting Software Feature vs Feature View functionality from the 2D Clusters view is available in the Feature vs Feature view including the point selection zooming and contour sorting functionality To display the Feature vs Feature view Select the View Feature vs Feature menu command or press the 88 button on the toolbar The Feature vs Feature view can operate in one of several modes which differ in the combinations of features that are shown in the plots You can select different display combinations via the context menu that is available by holding down the ALT key and right clicking in the view The modes are e Show All Active Feature Combinations shows every permutation of the currently Active Features This is the default mode for non Tetrode Data Types Generally the number of permutations is large enough that several pages are required to show all of the plots To step through the pages use the Next gt gt and lt lt Prev buttons on the top of the screen or the n Next or P Previous keys Feature vs Feature Next gt gt e Show PC1 4 Combinations shows the 6 permutations of the first 4 Principal Component projections PC1 vs PC2 PC1 vs PC3 PC1 vs PC4 PC2 vs Version 4 0 81 3 Views and Displays PC3 PC2 vs PC4 and PC3 vs PC4 In this mode press the nN or P keys to go to the next or previous channel as in the 2D Cluster
383. the Reset button A no B 2D Clusters AG PCA By default Offline Sorter Software plots the first 3 principal component eigenvectors in the main portion of the view Each different color corresponds to an eigenvector eigenvalue pair Offline Sorter Software calculates a maximum of 8 principal components eigenvalues eigenvectors All 8 eigenvectors can be displayed by selecting Show All 8 Eigenvectors from the right click menu The width of the line used to plot the eigenvector is proportional to the magnitude of the corresponding eigenvalue A bar chart graph of the eigenvalue magnitudes is shown in the upper right corner by default The graph can be moved by clicking on the interior of the plot and dragging it to a new location and it can be resized by clicking on the border and dragging The eigenvalue bars are scaled for Version 4 0 89 3 Views and Displays display in the graph such that the first eigenvalue takes up 90 of the range This graph is sometimes referred to as a Scree Plot The magnitude of an eigenvalue for a principal component eigenvector relative to the total of all eigenvalues summed represents the fraction of the total variance between waveforms that has been captured in that PCA dimension In the above screenshot it is clear that the first two principal components capture the vast majority of the variance So there is likely to be very little discriminatory power in the higher principal compone
384. the Timeline view CMR Common Median Referencing Very similar to CAR except instead of using the arithmetic average of the i th samples from each of the N input channels the median value of the i th samples from the N input channels is used instead This can in some cases result in better rejection of outlier samples from noisy channels With this method a new continuous Source named CMRMon that contains a single channel named CMRMon is created The remainder of the bottom portion is used to select the channels that will be used as input to the CAR or CMR calculation The Source droplist selects which continuous Source contains the channels that will be averaged If all channels in the Source are to be used in the CAR CMR calculation select the Use all channels in Source radio button or if only certain channels in the selected Source are to be used select the Use selected channels radio button and check the individual channels within the list 5 5 12 Waveforms Up sample Continuous Data Select this menu item to upsample the data in a continuous channel see Section 4 2 3 Specifying Up Sampling on page 114 This menu item is not available unless the current channel has continuous data 5 5 13 Waveforms Filter Continuous Data Select this menu item to apply a low cut digital filter to the data in a continuous channel Section 4 2 5 Selecting a Digital Filter Low cut or High cut on page 118 This m
385. the dialog that is displayed when Properties is selected from the context menu 3 18 Rasters View The Rasters view displays color coded tick marks that correspond to the times of unit firings which is similar to the Timeline view but the Rasters view does so for all channels simultaneously 92 Offline Sorter Offline Spike Sorting Software Rasters View To display the Rasters View Select the View Rasters menu command or press the E button on the toolbar WR ORM MOUE UNI eC Ta ee Fl ial a eta Bs PY UE UA OLANN NENN te Zoom Out Zoomin lt In addition the Rasters view can show the times when Events occur The Events can appear either as tick marks on separate raster lines or as lines overlaid across all rasters The Rasters view can only display the timestamps for channels that have been loaded into memory When a channel is loaded its timestamps are available for display To load all timestamps for all channels into memory select Load All Rasters from the right click context menu For some data files including PLX and PL2 the timestamps for all channels when the file loads can automatically be loaded by checking the Pre Load all Timestamps checkbox in the General tab of the Tools Data Import Options dialog However if this option is checked Offline Sorter Software uses more system memory and it slightly increases the time it takes to load the data The Rasters
386. the ellipsoid can be changed by pressing the 1 and o keys for fade in fade out while the 3D Clusters view has input focus The following screenshot is the same as the previous screenshot except the 1 key has been repeatedly pressed until the ellipsoids became totally opaque Run Valley Seeking Bes Delete Selected Unit E Info E 2D Clusters B 3D Clusters 68 Offline Sorter Offline Spike Sorting Software Surface View Note If enabling the ellipsoid display does not cause the ellipsoids to appear press the I key repeatedly as it is possible that the ellipsoids are there but are completely transparent 3 8 Surface View The Surface view displays the same density histogram described in Showing Dots or a Density Histogram on page 61 except that the histogram appears as a 2D surface in a 3D view as shown in the following illustration The control for the 3D viewpoint in the Surface view is the same as the control for the 3D Clusters view which is described in the preceding section To control the Density Histogram the same Density Histogram Settings dialog box can be used which appears on Showing Dots or a Density Histogram on page 61 To display the dialog box from the shortcut right click menu select Density Histogram Settings or press the D key The color of the surface above each histogram bin is a blend of the unit colors of the waveforms in that bin For example if 60 of the waveforms in a bin are
387. the first data channel into memory immediately after opening a data file You enable this feature by checking the Automatically Load First Channel after Opening a File checkbox in the General tab of the Tools Data Import Options dialog Version 4 0 111 4 Spike Sorting Step by Step To select a particular data channel 1 Click on the Channel drop list box in the Control Grid shows None in the illustration that follows 2 Select the channel from the drop list Range from s orin the Channels view of the Info Grid Views double click on the row of the desired channel S ae ae wersem unor Umra umro ome 112 Offline Sorter Offline Spike Sorting Software Waveform Acquisition From Continuously Recorded Data The Timeline view will then show the channel s data Control Displays Z Center Selected Zoom 10 5 00 mV Th 237 1183pV Detect Waveforms Options Up Sampling None 5 Frequency 40 Low cut Fitter None M Scroll Range 10 0000 0 0000 Zoom Out 3 To move quickly to the next or previous channel in the list of channels click Select Next Channel or Select Previous Channel The following toolbar buttons can also be used an 4 Offline Sorter Software maintains a history of previously opened channels To revisit a previously opened channel click Edit Previous Channel in History or click Edit Next Channel in History The following toolbar buttons can also be
388. the principal components in the 2D Clusters view as follows in the right side illustration 2500 my 800 usec The variance within waveform groups can frequently be reduced and the separation between waveform groups enhanced by aligning the waveforms at their minimum or maximum values Note If spikes are extracted from continuous data that is present in the data file better results are usually obtained by using the Align During Extraction feature To set the alignment criteria 1 Select the Waveforms Align menu command Or select the Tools Align Waveforms menu command Version 4 0 139 4 Spike Sorting Step by Step 2 Then select the desired alignment method G Which Channels Align Waveforms On First Local Maximum after Sort Start First Local Minimum after Sort Start First Local Extremum after Sort Start Global Maximum between Sort Start and Sort End Global Minimum between Sort Start and Sort End First Local Extremum after Pre T hreshold Units Ignore unit assignments when aligning waveforms Align waveforms within each unit independently to improve timestamp resolution use after units have been sorted Only align waveforms assigned to the currently selected unit For stereotrode tetrode data align waveforms in each electrode independently v Adjust timestamps Maximum Alignment Shift in clock ticks 2 Actions After Alignment
389. the scan results For more information see Section 3 19 Scan Graph View on page 94 Sort Quality vs Time Segment Graph View The Sort Quality vs Time Segment Graph view shows how the sorting quality statistics changed as a function of time throughout the duration of the file See Section 3 20 Sort Quality vs Time Segment Graph View on page 97 Threshold Scan Graph View The Threshold Scan Graph view creates and displays a graph of the number of extracted spikes and the signal to noise ratio as a function of the threshold This can be helpful in deciding where to set the threshold for extracting spikes from continuous data See Section 3 21 Threshold Scan Graph on page 97 Overlaps Analysis View The Overlaps Analysis view can detect and rescue overlapping waveforms If a second spike occurs within the collection window of a previous spike the two waveforms are said to overlap These overlapping waveforms consist of superimposed spikes from different units and generally show up as outliers because they do not have spike shapes similar to any single unit For cases where spikes have been extracted from continuous data within the Offline Sorter Software the Overlaps Analysis view can mathematically remove the superposition and thus separate each overlapping spike into distinct spikes See Section 3 22 Overlaps Analysis View on page 99 Waveforms Legacy View The Waveforms Legacy view is provided only for specia
390. the waveform peak heights The dashed white line indicates zero and dashed blue lines indicate the 3 sigma noise level The solid red line marked with a orange arrow below indicates the selected threshold For the Peak Heights histogram only the first buffer of continuous data see Section 6 11 3 Buffer Sizes on page 326 is scanned to find the peaks A positive peak in this case is defined as a sample where the two samples surrounding it on either side are less than the sample value likewise a negative peak is where a sample is less than more negative than 2 samples on both sides of the peak sample The number of peaks found at every different peak height amplitude is tallied in bins to form the Peak Heights histogram 74 Offline Sorter Offline Spike Sorting Software Waveform Detection View 4 2D Clusters 3D 3D Clusters Surface Peak Histogram Timeline Control Displays Center Selected Zoom 1 0 5 00 my Thr 4 22 8 1140 pi l Detect Waveforms Options Up Sampling None m as Frequency Low cut Filter None x Scroll Range 10 0000 0 0000 Zoom Dut Zoomin lt 3 11 1 Threshold Calculation Offline Sorter Software uses the following algorithm to calculate the noise sigma that are plotted as vertical lines on the Peak Histogram and are used in the Waveform Detection dialog see Section 5 5 14 Waveforms Detect
391. this menu 5 10 Run Menu The Run menu contains entries for quickly and easily launching other software E Pledtil NeuroExplorer See Appendix A 3 2 Run Menu on page A 13 for a description of the items in this menu 272 Offline Sorter Offline Spike Sorting Software Help Menu 5 11 Help Menu The Help menu contains entries for providing information about Offline Sorter Software Help Topics Eo Quick Key Reference Quick Batch Reference P About Offline Sorter 5 11 1 Help Topics Click Help Topics to open the on line version of this manual 5 11 2 Quick Key Reference Click Quick Key Reference to open the following window 2D Cluster View Lef t Click Select nearest point lt Right Click Displays context menu Right Click Drag Zoom the image both axes together Ctrl Right Click Drag Zoom the image each axis independently Shift Left Click Drag Pan the image Ctrl Left Click Drag Start drawing contour for new unit Ctrl Left Click Set cluster center K Means StdEM sorting only Double Left Click Set cluster center K Means StdEM sorting only LG Ae Increase decrease contrast for density histogram display Toggle linear logarithmic range mapping for density histogram disple Cycle smoothing method for density histogram display Display density histogram settings dialog Toggle ellipse display for units Next channel Previous channel Esc Cancel add remove invalidate mous
392. ting or clicking Recalculate the Principal Components To re enable the display click to clear the Do not show the Recalculate Principal Components dialog checkbox on the Feature Calc tab of the Tools Options window 298 Offline Sorter Offline Spike Sorting Software Details of the Sorting Algorithms 6 4 2 Voltage at Specific Slices in Time To view waveform voltages at specific points in time on the X Y or Z axis of the Clusters views e Select Slice 1 Slice 2 Slice 3 or Slice 4 for the X Y or Z axis using the Drop Down List Boxes on the Toolbar e Adjust the time values for Slice 1 Slice 2 Slice 3 or Slice 4 in the Control Grid When visible you can also drag the Slice 1 Slice 2 Slice 3 and Slice 4 lines into position with the mouse on the Waveform display e Right click the Clusters view and select Redraw in the pop up menu qo y X Axis Slice 1 Y Axis Slice 2 ai e To update the Clusters view immediately when any of the slice n values are changed select the Redraw Cluster Display Immediately when Slice Position Changes in the Display tab under the Tools Options dialog box e By default each channel remembers its slice positions independently To have the same slice positions used across all channels click Use Same Slice Positions for all Channels in the Feature Calc tab under the Tools Options dialog box 6 5 Details of the Sorting Algorithms 6 5 1 Feature Space Used for Sorting
393. ting the Sort Range on page 142 Select a feature space in which to perform sorting See Section 4 4 6 Selecting Features to Use For Sorting on page 143 Select units using one of the supported spike sorting methods See Section 4 5 Spike Sorting on page 143 4 For each sorted unit Clean the selection if necessary See Section 4 6 3 Cleaning the Selection on page 187 If relevant rescue overlapping waveforms See Section 4 6 5 Rescuing Overlapping Waveforms on page 194 Inspect individual waveforms if necessary See Section 4 7 1 Waveform Inspection on page 198 5 Save the timestamps as a NeuroExplorer data file See Section 4 8 2 Exporting to a NEX File on page 206 or save the timestamps and waveforms in a data file See Section 4 8 1 Saving Timestamps and Waveforms on page 205 4 2 Waveform Acquisition From Continuously Recorded Data 4 2 1 Opening the Digitized Data Files Offline Sorter Software can sort waveforms from files containing continuously digitized data such as that generated by other vendor s files or from other binary files that contain multiplexed A D values recorded from several channels For more information about reading and importing data files see Section 5 1 2 File Import on page 221 To open a continuously digitized Plexon data file ta press File Open toolbar button or select File Open menu command To open a continuously digitized data
394. tion calculation The MAD calculation involves finding the median of the absolute deviations from the data s median value When used to find the MAD between a waveform and its unit template waveform the MAD value is calculated on the quantity d w t MAD median abs d m where m median d That is first find m which is the median of all the differences between each waveform and the unit template Then the MAD is the median of the absolute values of the residual between each d and m Version 4 0 301 6 Reference Topics Finally the modified standard deviation is found by multiplying the MAD value by a scaling constant sman MAD 1 4826 This modified standard deviation is generally used in place of the normal standard deviation for subsequent calculations 6 5 4 Adaptive Templates Algorithm The adaptive template algorithm behavior is controlled by two parameters e the Weight e the Update Threshold which can be set on the Tools Options Templates tab Adaptive Templates Weight 0 01 Threshold for Template Update 10 To adapt the templates Offline Sorter Software will process the spikes in the file in time order As it proceeds it will maintain both the current template waveform being used for sorting t i and an average waveform s i for each unit where i indexes the sample within the waveform The average waveform s i starts out equal to the initial template waveform t i at the
395. togram runs from zero to ISIHistMax The graphs also show the total number of spikes for each unit Larger printable versions of these Interspike Interval Histograms can be displayed in the Section 3 16 ISI Histograms View on page 90 42 Offline Sorter Offline Spike Sorting Software Units View The Unit Display shows the percentage of spikes with Interspike Intervals less than the Refractory Interspike Interval in red which also includes a red bar in the upper portion of the display The values of ISTHistMax and Refractory Interspike Interval can be changed in the Control Grid The Refractory Interspike Interval value can also be set by using the Refractory Period tab on the Tools Options menu command Selecting Units Units in the Units view can be selected by left clicking on the corresponding template Offline Sorter Software draws a solid highlighted box and a colored background on the box corresponding to the currently selected unit For purposes of selecting units the Unsorted unit behaves just like the actual sorted units When the Waveforms view setting in the Control Grid is Sel Unit s selecting units by clicking in the Units view is a convenient means to quickly see the differences and similarities between units Offline Sorter Software uses the concept of selecting a single primary unit and multiple secondary units The primary unit is the unit that is affected by operations such as Delete Selected Unit
396. trode Waveforms checkbox in the Tools Data Import Options Plexon dialog controls what happens when the waveforms from all 2 or all 4 channels are not found in the data Version 4 0 291 6 Reference Topics file The incomplete waveform is added with zeros filling the missing channel waveform s but that incomplete waveform is marked as being invalid when the checkbox is checked the default In all data file formats for which Offline Sorter Software combines multiple individual raw channels to form stereotrode and tetrode channels Offline Sorter Software uses the characteristics of the first raw channel in the stereotrode tetrode gouping as the characteristic for the overall stereotrode tetrode channel That is the channel name number of units filtering parameters and per waveform unit designation are always taken from the first raw channel in a stereotrode or tetrode channel However there are two exceptions the Threshold value is taken from each individual raw channel as Offline Sorter Software supports having a different threshold for each channel making up a stereotrode tetrode channel and the gain Since Offline Sorter Software has only a single gain value for the full stereotrode or tetrode channel it will always use the first raw channel s gain as the stereotrode tetrode channel gain But for Plexon plx and pl2 files Offline Sorter Software has a feature to compensate for differing gai
397. ture of the waveform across all electrodes The threshold by default is the same for all electrodes in the channel To adjust the threshold for each electrode independently click the Detect Waveform Options button to open the Waveform Detection Options dialog box then click For stereotrode tetrode data allow an independent threshold for each electrode In the preceding illustration the purple highlighting box shows which electrode is current for the purpose of adjusting the independent threshold 6 3 3 Sorting Stereotrode and Tetrode Spike Waveforms By default Offline Sorter Software analyzes stereotrode and tetrode spike data by concatenating the individual waveforms from each electrode in the tetrode or stereotrode group into a single waveform The resulting concatenated waveform is twice for stereotrode or four times for tetrode as long as each individual waveform The Waveform Display shows the concatenated waveform with vertical white lines separating the waveforms from each individual electrode channel within the group The Timeline view represents the waveform group arranged vertically indicating that they were acquired at the same point in time Version 4 0 293 6 Reference Topics Waveform Display 1556 9340 Zoom Out Zoom You can then apply all cluster display and sorting features to the concatenated waveforms Offline Sorter Software includes sorting features that calculate values on the per electrode wa
398. u Options The two selected units will be swapped e g the waveforms that were sorted into Unit a become sorted into Unit d and vice versa 5 7 6 Units Invalidate All Waveforms in Selected Unit Selecting this menu item will invalidate all waveforms that have been sorted into the currently selected Unit This same function is also available from the Waveforms menu 5 7 7 Units Add Waveforms to the Selected Unit Selecting this menu item will put the Waveforms and Clusters views into the mode where the user is expected to cross on the Waveforms view or circle on the Clusters views the waveforms that should be added to the currently selected Unit This is equivalent to pressing the m wfs button on the per view toolbars or the button on the main toolbar 5 7 8 Units Remove Waveforms from the Selected Unit Selecting this menu item will put the Waveforms and Clusters views into the mode where the user is expected to cross on the Waveforms view or circle on the Clusters views the waveforms that should be removed from the m Remove Wis button on the currently selected Unit This is equivalent to pressing the per view toolbars or the button on the main toolbar 5 7 9 Units Remove All Except Selected Waveforms from Selected Unit Selecting this menu item will put the Waveforms and Clusters views into the mode where the user is expected to cross on the Waveforms view or circle on the Clusters view the wave
399. uality metrics Scan Number of Waveforms To Use In PCA This scan type re runs the PCA using only the first N waveforms where N is the step value Then Offline Sorter Software recomputes the sort quality metrics Although the unit assignments are not altered re running the PCA changes the positions of points in PCA based feature space and so the sort quality metrics can change Version 4 0 181 4 Spike Sorting Step by Step This feature is useful to examine the sensitivity of sorting results to different PCA results e Scan Random Seed for K Means This scan type re runs the K Means after randomly distributing the initial cluster seed points across the region in feature space spanned by all the waveforms The scan value is the actual seed used in the random number generator so entering start stop step values of 1 0 10 0 and 1 0 will result in 10 steps the first using a random number seed of 1 0 the second using 2 0 etc up to a random number seed of 10 0 Note that for this scan method a new line appears where the number of K Means units can be fixed to a specified value So if a value of 3 is entered at each step 3 randomly positioned seed points will be created resulting in 3 clusters being created Thus from step to step the only difference between the sorting is the random placement of the initial cluster centers e Scan Random Seed for Std E M This scan is exactly like the random seed scan for K Means except tha
400. uld be combined with the last full duration time segment in the file if the amount of leftover time is less that the specified number of seconds or if it should become a new shorter segment To divide up the file evenly into equal duration time segments select the second radio button and specify the number of segments The third radio button allows the quick creation of a single time segment covering the specified time span all other pre existing time segments are deleted The fourth radio button allows adding arbitrary time segments onto the list of existing time segments Complex series of time segments can be built up in this way For the first two options generic names will be automatically generated for the new time segments For the second two options a name can be optionally specified for the new time segment if it is left blank a generic name will be automatically generated 244 Offline Sorter Offline Spike Sorting Software Waveforms Menu However the time segments were specified Offline Sorter Software calculates statistics and fits a 2D ellipse to the points in every segment The Clusters vs Time view can plot these 2D ellipses as semi transparent elliptical cylinders that cover the time range of the segment i Clusters Vs Time oe When plotted in this fashion the elliptical cylinders for each unit stack up into a tube shape these tubes are called PCSTs PCA Stability Tubes These tubes can be show
401. umber of channels with spikes The Invalidate Artifacts tool completely ignores any channels that have 0 spikes in them and assumes they are inactive or disabled channels Because artifact waveforms can cross different thresholds at slightly different times different threshold settings on different channels effect the timestamps of artifacts The time width setting provides a way to bracket timestamps so that artifacts are still recognized with different thresholds You can set the channel presence criterion to take into account the fraction of channels that contain high quality analyzable data as opposed to occasional noise triggers Using automatic artifact invalidation 1 Select which spike Source will have artifacts removed If there is only one possible choice a single spike Source it is automatically selected 2 Click File Invalidate Cross Channel Artifacts The Invalidate Artifacts window opens Ei Invalidate Cross Channel Artifacts This tool will invalidate artifacts that are present on several channels within a spike Source The artifacts are identified only by their time coincidence within the artifact width parameter across channels Spike Source SPK Chs 1 4 Maximum width of artifacts 1 ticks 25 0 psecs To be an artifact time coincident waveforms must be present on atleast 90 of the channels with spikes CK cancel _ D 3 In the Maximum width of artifacts box type the maximum time
402. umn of the appropriate grid based window A 3 7 Undo Plexon applications provide multiple undo levels To undo an operation on the Edit menu click Undo or click the Undo button on the toolbar You can undo operations that change the contents of the project file but you cannot undo operations that change the user interface options or colors Version 4 0 Appendix A Navigating the Plexon UI A 4 Customization Although the menus and toolbars offer a rich set of commands and functions that should meet the needs of most Plexon customers the Customize dialog box also allows you to customize several areas of the interface should the need ever arise This section describes the uses of the Customize dialog box To open the Customize dialog box from the Tools menu select Customize The Customize dialog box contains several tabs The illustrations below show the Customize dialog box from CinePlex Editor and displays five tabs Toolbars Commands Keyboard Run Menu and Options Although the content of the tabs will vary according to the application the functional operation of each tab is respectively the same across all Plexon software applications A 4 1 Toolbars Customization An illustration of the Toolbars tab follows Customize Toolbars Commands Keyboard Run Menu Options Toolbars Me a Standard MIEdit _ Animate Looping Find Fre M Views e Toolbars This box contains a list of the toolbars
403. unit template by crossing waveforms then you can adjust the tolerances and the template itself by dragging on screen handles Note The Lines and Bands sorting methods function the same way in Offline Sorter Software as in the OmniPlex D System Semi automatic sorting methods The Semi automatic sorting methods generally require specifying cluster centers and therefore implicitly indicate the number of clusters and then the algorithm assigns waveforms to the clusters The Template method requires picking an existing waveform to serve as the template for a unit Offline Sorter Software then adds other waveforms to the unit based on their similarity in the least squares difference sense to the template unit with a user defined tolerance The K Means method is a well known iterative algorithm that assigns each waveform to one of the user specified cluster centers based on Euclidean distance in feature space Then it recomputes the cluster centers and it repeats the process until no more waveforms change units The Standard E M Std E M method is a variation of the Expectation Maximization algorithm The Expectation Maximization algorithms in general fit a mixture of Gaussians to the point densities in feature space by varying the normal distribution parameters means covariances to maximize a likelihood function This algorithm uses the user specified center points to start the search The Semi automatic sorting methods are also capa
404. ures can be made active so that they appear in the X axis Y axis and Z axis feature drop lists in the toolbar See Axis Selection under Section 3 7 2 Clusters Views Common Functionality on page 54 294 Offline Sorter Offline Spike Sorting Software Features Available for Sorting In the following w t w 1 w n is the waveform n number_of_points_in_waveform pl t p1 1 p1 n is the first principal component vector p2 t p2 1 p2 n is the second principal component vector p3 t p3 1 p3 n is the third principal component vector x represents a sum over t The available features are e PCi The waveform projection onto the first principal component PC1 Xp1 t w t e PC2 The waveform projection onto the second principal component PC2 Xp2 t w t e PC3 The waveform projection onto the third principal component PC3 Xp3 t w t e PC4 PC8 The waveform projections onto higher principal components e Slice 1 The waveform height at a selected position in time You can select the time position in the Control Grid or the Waveform display Slice 1 w i the waveform voltage at time t i e Slice 2 The waveform height at a selected position in time You can select the position in the Control Grid or the Waveform display Slice 2 w j the waveform voltage at time t j e Slice 3 The waveform height at a selected position in time You can select the position in the Cont
405. used Ed bh The default setting is the Raw Signal detector in which case the Waveform Detection view shows a histogram of the logarithm of the Peak Heights The white lines on the displays indicate the current threshold setting Version 4 0 113 4 Spike Sorting Step by Step 4 2 3 Specifying Up Sampling Offline Sorter Software can increase the sampling frequency of continuous data channels by interpolating new samples between the original samples This is useful in situations where continuous data is sampled at 20 KHz in the same file with spikes digitized at 40 KHz Spikes cannot be extracted from the 20 KHz channels unless and until they are up sampled to the spike frequency of 40 KHz Note that the new up sampling frequency must be 1 a multiple of the original sampling frequency and 2 less than the spike frequency for the file Two different interpolation methods are available Linear and Spline Spline interpolation generally gives better results but is limited to doubling the original frequency of the continuous data Linear interpolation is more flexible in that it can create any multiple of the original frequency but the result is of somewhat lower quality To enable up sampling select Up sample Continuous Data from the Waveforms menu Which Channels Sets up sampling for Source FP Chs 17 32 Upsampling Parameters Interpolation Method Linear New Frequency 2x to 2000 Hz apply Lox cance
406. usters 3D Clusters Surface Clusters vs Time Templates vs Time Waveform Inspection Waveform Detection Continuous Frequency Spectrum hin Feature vs Feature Sorting Summary PCA Results Histograms Cross Correlograms Rasters Sort Quality vs Time Segment Graph Threshold Scan Graph Overlaps Analysis Waveforms Legacy Animate Control Grid View Sources Channels Channel Parameters File Summary Time Segments Sort Details Events Stats PCA Scan Templates Adaptive Templates History Channel Map Messages The Control Grid is used to select and set values for important control parameters and viewing options See Section 3 3 Control Grid on page 38 Units View The Units view shows the templates average waveforms for each sorted unit plus the remaining unsorted waveforms Click in this view for the fastest way to 22 Offline Sorter Offline Spike Sorting Software Overview select the current unit The graphs at the bottom of the view display the histograms of interspike intervals See Section 3 4 Units View on page 42 Waveforms View The Waveforms view shows the waveforms for the selected channel which are all superimposed The mouse may be used to add and remove waveforms from units in this display For more information see Section 3 5 Waveforms View on page 45 Timeline View The Timeline view shows the waveforms and or continuous data as a funct
407. ution in 2D or 3D space The p value is the probability that an error is committed by rejecting the null hypothesis Therefore a small p value indicates that each of the unit clusters has a statistically different location in 2D 3D space and that the clusters are statistically well separated After clusters are selected in 2D or 3D cluster space you can view the resulting F and p values derived from the MANOVA test in two and three dimensions in the Stats Spreadsheet in the Info Grid Views which is detailed on page 25 and in the Clusters views see Section 3 7 2 Clusters Views Common Functionality on page 54 6 6 1 1 Some details on the MANOVA calculation In the jargon of MANOVA the MANOVA number of groups is our number of units and the MANOVA dependent variables DV are our feature vectors e g principal components This analysis is that of a single Factor experiment with the Treatment being the unit assignments The MANOVA F statistic is calculated using a ratio of determinants to get Wilk s lambda followed by Rao s method for converting Wilk s lambda into an F value See Applied Multivariate Statistics for the Social Sciences 2nd Edition by James Stevens page 191 for more details An unweighted MANOVA is performed in that all groups regardless of the number of waveforms they contain contribute equally to the F statistic calculation so as not to bias the MANOVA calculation toward rapidly fir
408. valid size for the header e Once the continuously digitized data starts after the header it cannot have any kind of block structure to it it must contain just A D values arranged in sweeps each sweep contains a single A D value from each channel e Each A D value is stored in a signed 16 bit word regardless of the A D conversion resolution If the Swap Bytes checkbox is checked Offline Sorter Software interprets the 16 bit words in big endian MSB first format instead of the default Intel standard little endian format To import a binary file from the main menu select File Import Binary File with Continuously Digitized Data The Import Binary Digitized Data File dialog box appears 224 Offline Sorter Offline Spike Sorting Software File Menu Import Binary Digitized Data File File To Import C PlexonD ata Plexon ddt 4channels40khz ddt Import Parameters Number Of Channels Data Offset from the start of the file bytes Digitizing Frequency Hz 4 D Converter Resolution bits 12 Maximum Voltage rm 5000 Swap Bytes Load from ofi File Save to ofi File Load Using Companion ofi File Cancel Select a file then enter the required information in the Import Parameters area Click OK to close the dialog box and import the file Note You can click Save to ofi File to save the choices of Import Parameters to a file that by convent
409. variables can be imported as time segments A time segment is created for each occurrence of each different NeuroExplorer Interval 5 1 2 9 Neuroshare Native Import for Neuroshare native NSN files is available only via the Neuroshare DLL which can be obtained from www neuroshare org You must download this DLL file and use the Neuroshare tab of the Tools Data Import Options dialog box to set the name and location of this DLL in the Neuroshare Native nsn File Format section Version 4 0 223 5 Menu Options 5 1 2 10 Panasonic MED Import for Panasonic med files is available via a native loader This format contains only continuous data samples 5 1 2 11 R C Electronics Support for R C Electronics files is available only via the Neuroshare DLL which can be obtained from R C Electronics see www neuroshare org One must download this DLL file and use the R C Electronics tab of the Tools Data Import Options dialog box to set the name and location of this DLL 5 1 2 12 Generic Binary Continuous File Import Offline Sorter Software can sometimes import a raw binary file that consists of continuously digitized samples Note The binary file is expected to conform to the following specifications e The data file can contain a fixed size header block at the beginning of the file The size of this header block must be known and entered into the dialog so that this part of the file can be skipped over Zero is a
410. ve described sort quality metrics are applied to the entire channel a single number attempts to describe the sort quality for all units in a channel Offline Sorter Software can also calculate some sort quality metrics that are defined on a unit by unit basis That is for a per unit sort quality metric a single quality number is calculated and reported for each unit where that number attempts to describe some aspect of the quality of that unit Offline Sorter Software supports calculating two per unit sort quality metrics called L Ratio and Isolation Distance Both of these values are calculated in feature space and so can be calculated both in 2D and in 3D feature space L Ratio is a measure of the amount of noise near the unit where noise in this case means waveforms that are not assigned to the unit under consideration A low value of L Ratio indicates that the unit is well separated and not contaminated by nearby spikes that are not assigned to the unit The value of L Ratio is essentially a weighted sum over waveforms not assigned to the unit under consideration with the weight determined by how close the foreign waveform is to the unit Version 4 0 309 6 Reference Topics center Foreign spikes that are close to the unit center hurt more and contribute more to the value of L Ratio than those further away Isolation Distance is a measure of how distant the non unit spikes are from the spikes in the unit under co
411. ve template threshold will cause the algorithm to be eager to adopt new templates whereas larger values will make the algorithm more reluctant to change templates 6 5 5 K Means Algorithm The K Means algorithm is described in any pattern recognition textbook The algorithm runs in feature space and requires starting positions for the clusters and thus implicitly the number of clusters to be specified before the algorithm begins Algorithm outline 1 The user first sets initial cluster centers 2 Each point is assigned to the cluster whose center is nearest using a simple 2D or 3D Euclidean distance 3 Cluster centers are then recalculated using the points assigned to each cluster using a simple center of gravity method Steps 2 and 3 are repeated until the cluster centers no longer change When the K Means algorithm converges an outlier removal algorithm runs The standard deviation of the distances from each point to its cluster center computes Any point that is more than a user specified constant OutlierThreshold times this standard deviation away from its cluster center is removed from the cluster References Webb Andrew Statistical Pattern Recognition Second Edition John Wiley and Sons 2002 and many others 6 5 6 Standard E M Algorithm The Expectation Maximization algorithm is a widely known iterative parametric approach that fits a normal mixture model a mixture of several Gaussians to the available da
412. ved TPL file 3 perform either template or Contour sorting on the spikes 4 8 5 Saving and Restoring Scan Results After a scan has been performed the results of the entire scan including the sorting done at each step can be saved to a file and later recalled To save the results of a scan to a file select Save Scan Results to a File from the right click menu on either the Scan view or the Scan Graph view To recall a previously saved scan select Restore Scan Results from a File When saving or restoring a scan a filename in a standard format is suggested with the suggested format being 214 Offline Sorter Offline Spike Sorting Software Saving the Sorting Results lt filename gt _chan lt NNN gt scan where lt filename gt is the name of the file currently being processed and lt NNN gt is the current channel number A feature in Offline Sorter Software exists to load automatically a SCAN file for a channel if it exists in the same directory as the data file and is named according to the above format When the companion SCAN file is loaded it makes the complete results of the scan available for inspection To enable this feature check the Automatically Load Companion SCAN Files if they exist checkbox on the Tools Data Import Options General tab If the Warn when loading sorting results from a SCAN file checkbox is checked the following dialog will appear whenever a companion SCAN file is found f
413. veforms Waveform Length microseconds Prethreshold Period microseconds 200 Dead Time microseconds Note that OfflineSorter does not support different waveform lengths for different channels If you extract waveforms and then change the waveform lenath all the extracted waveforms will be erased _ For stereotrode tetrode data allow an independent threshold for each electrode Detector Detecto Raw Signal z Use Dual Thresholds Always set Thr 2 Thr Continuously Sample Raw Signal l Adjust Gains Alignment During Detection Raw Signal Only C Detect Now Se etect Now After threshold crossing align to the largest peak Same Side as Threshold Either Side Cancel This is because otherwise the waveform detection code may well detect the threshold crossing from an overlapping waveform within the time window of the first waveform This will interfere with the Overlaps Analysis and will in effect lead to double counting of overlaps each of the waveforms that already overlap in time because of the Dead Time being too short could be again split into two overlapping waveforms by the Overlaps Analysis algorithm Also do not use Alignment During Detection or manually perform Waveform Alignment as this will also interfere with the results of the Overlaps Analysis Sort Units Conservatively Overlaps Analysis requires sorted units as a starting point When sorting s
414. veforms instead of the concatenated waveforms e g Peak EL 1 is the peak value of the waveform on the first electrode only Also ratios can be formed from per electrode features e g Peak EL 1 EL 2 is the peak value on electrode 1 divided by the peak value on electrode 2 Stereotrode and tetrode data formats in Plexon PLX and PL2 DataWave UFF and Neuralynx files are automatically detected when the file is opened 6 3 4 Disabling Electrodes With Stereotrode Tetrode data an electrode or wire can break or otherwise just produce noise or artifacts You can choose to ignore individual electrodes within a stereotrode tetrode by clicking open the Electrode Enable drop list in the Control Grid and unchecking the checkboxes The Electrode Enable entry in the Control Grid will display N A if single electrode data is loaded into Offline Sorter Software Invalid w f Show Electrode Enable 1 2 3 4 iv lee oe Electrode 2 A Electrode 3 Electrode 4 When an electrode is disabled a red box with the word Disabled will appear in the space where that electrode is being displayed in the waveform display Disabled electrodes do not contribute to PCA calculations or to calculations of any other Feature values for the waveforms 6 4 Features Available for Sorting You can choose each dimension of the 2D or 3D feature space used in Offline Sorter Software from the list of available features Any of the following feat
415. veral features for each axis The features available for assignment to the axes in feature space include e Projections onto principal components PCA e Waveform heights at chosen times slices e Peak height valley height peak valley difference widths e For stereotrode and tetrode data per electrode and ratio between electrodes features Thus the waveform can be represented by the point x y in a 2D feature space or x y z in a 3D feature space where the x y and z axes can be chosen from the available features In the 3D view of the feature space points it is possible to navigate the eyepoint through the 3D space to get a better idea of what the data set looks like and to find a vantage point where clusters are best separated After the desired perspective in 3D space is achieved the sorting methods can be applied For a detailed description of the features see Section 6 4 Features Available for Sorting on page 294 Manual sorting methods Manual spike sorting methods allow full control of the sorting process and it is possible to indicate manually which waveforms should be assigned to each unit With the Waveform Crossing method the units are manually selected by using the actual waveforms Crossing a bundle of waveforms with the mouse pointer can specify the unit Using the Contours method a cluster is specified by drawing an arbitrary shape in feature space usually around a visible cluster Offline Sorter
416. view displays the mean template for each sorted unit See Section 3 2 11 Templates View on page 37 The Adaptive Templates History view is only relevant if sorting with adaptive templates was performed Then it displays a time stamped history of when the templates were changed along with the numerical values for the 26 Offline Sorter Offline Spike Sorting Software Info Grid Views new templates See Section 3 2 12 Adaptive Templates History View on page 37 The Channel Map view shows the mapping between raw data channels from the data file and the channel number used by Offline Sorter Software This mapping includes the ability to combine raw channels to form stereotrodes and tetrodes See Section 3 2 13 Channel Map View on page 37 The Messages view displays a list of significant occurrences within Offline Sorter Software It is useful for debugging problems or reminding oneself of what operations were previously executed See Section 3 2 14 Messages View on page 37 3 2 1 Sources View e fe oe pos pea 1 pono pe aee oe ma e veele 6 Single bitevents Event 11 42 Other events CinePlex Data SPK_SPKC 2500 000 32 8 1 32 2500 000 2500 000 2500 000 Spike 1 16 40000 Cont 17 32 40000 Cont 33 48 40000 Cont 49 64 1000 Event 3 10 Event 43 45 Event 46 48 Spike 33 48 4 40000 Y iy iY Y iY Y iY Y iN 16 2500 000 32 8 1 32 The
417. w slide is added to the presentation Sending another Sorting Summary view to PowerPoint results in another slide being added to the same presentation PowerPoint does not save the presentation to disk select Save from the PowerPoint menu to save the presentation 3 14 3 Outputting for All Channels The File Print All Sort Summaries and File Send All Sort Summaries to PowerPoint iterate through all channels and print or export the Sort Summary for each channel to a single PowerPoint file or to a single print job 3 15 PCA Results View The PCA Results view graphically displays the results of the Principal Components Analysis PCA that runs on the waveforms The calculated principle component eigenvectors form a basis onto which waveforms can be projected in order to get the PC1 PC8 feature values A bar graph of the eigenvalues associated with each eigenvector is also displayed The same PCA 88 Offline Sorter Offline Spike Sorting Software PCA Results View information is displayed in table form in the PCA view of the Section 3 2 Info Grid Views on page 25 To display the PCA Results View Select the View PCA Results menu command or press the W button on the toolbar Note If this button does not appear on the toolbar select Toolbars tab under Windows Customize and select the line labelled View in the list box in the Toolbars tab Do not uncheck the checkbox just select the line Then click
418. width in ticks units of 25 0 us of the artifacts to be invalidated Note For example if the timestamps on two channels differ by this number of ticks or less the tool considers the spikes on the two channels to be simultaneous If enter 0 zero is entered the tool only invalidates artifacts with identical timestamps 4 In the of the channels with spikes box type the channel presence criteria in percent For example in the illustration in Step 1 the tool invalidates 136 Offline Sorter Offline Spike Sorting Software Preparation For Sorting artifacts of the specified width and with simultaneous timestamps that are present on 80 of the channels with spikes 5 To start the tool click OK 4 4 3 Examining Events Although not strictly relevant to spike sorting Offline Sorter Software provides the ability to examine the events in data files The Events view of the Info Grid Views lists the events present in the file by name and channel number with a Count of the number of times that events appears along with a Show checkbox and a display color for each EventCh Name Count Show Display Color EE 3 cok When the Show checkbox is checked for an event Offline Sorter Software plots each occurrence of that event as a line of the corresponding Display Color on the Timeline view and on the Rasters view so you can inspect the temporal relationships between events and nearby spikes In the Timeline view t
419. ws and Displays 3 6 Timeline View The Timeline view shows the data either continuously digitized or waveform segments in its proper temporal sequence This window can be used to e Specify an optional digital filter select a threshold and extract waveform segments for continuously recorded data e Control the temporal range of waveforms shown in the Waveforms view and Clusters views e Select individual waveforms left mouse click on waveform for viewing in the Waveforms view and Waveform Inspection view e Create Time Segments that encompass a set of waveforms 3 6 1 Stripchart Display in the Timeline View There are two sections in the stripchart display see the area with the black background in the image below The bottom section shows the continuous data and the top section shows the spikes for a channel The two sections are separated by a moveable splitter If there is no continuous data showing the spike section fills the entire stripchart section and conversely if there is no spike data showing the continuous data section fills the entire stripchart display Spike Ch 21 SPK21 Showin T Cont Ch 85 WB2T EJ Control Displays Center Selected Zom Mm 2 50 mV Thi 6 6 166 pV Detect Waveforms t t Low cut Filter None 4 pole Cutoff Freq 251 Hz Scroll Range 10 0000 0 0000 Zoom Out Zoomin lt m 48 Offline Sorter Offline Spike Sorting Software Timeline View Note A single ch
420. xplorer datafile If Offline Sorter Software is used to open a Spike Waveform data file e g a Plexon PLX or PL2 file or a data file from the DataWave or Neuralynx systems the resorted waveforms and spike times can be saved in a file of the same format or in a new PLX file The Offline Sorter Offline Spike Sorting Software Offline Sorter Inputs resulting spike files PLX PL2 NEX etc can then be analyzed with NeuroExplorer MATLAB etc A diagram of the typical analysis pathways and files related to the Offline Sorter Software follows As shown in the diagram Offline Sorter Software can work with PL2 files generated by the OmniPlex D System or with PLX files generated by either the MAP System or the OmniPlex D System NeuroExplorer i Data Analysis Sort Client Data file Data file OmniPlex PlexControl Template file Live recording Offline Sorter Data file DataWave from other 7 Multichannel system Systems 2 2 Offline Sorter Inputs Offline Sorter Software can open or import data files recorded from Plexon data acquisition hardware PLX and PL2 files and files from various other data acquisition hardware vendors In general these data files contain at least three types of data e Continuously recorded data With this type of data an analog signal is digitized at a certain rate and every digitized sample is stored in the data file Examples of this type of file
421. y Histogram only select Show Density Histogram from the right click shortcut menu To show both surfaces simultaneously with transparency select Show Both Density Histogram and Unit Fits To toggle the display through these settings press the F key When both surfaces appear together the two surfaces can be faded by pressing the I or O key or the Contrast slider in the Density Histogram Settings dialog box can be adjusted The Surface view can be used to perform Contour sorting When a contour sort in the Surface view is done think of the contour as being drawn on the base counts 0 plane Any existing contours are projected onto the surface and plotted as lines 3 9 Clusters vs Time View It is useful to examine and understand the time structure of when spikes occurred in the experiment The Timeline view provides this functionality while showing the spikes in waveform space but the Clusters vs Time view shows this information in feature space Examining the Clusters vs Time view can indicate at a glance just how spikes are distributed within the data file e g Unit A was Version 4 0 71 3 Views and Displays only firing for the first half of the experiment or that waveform shapes changed as a function of time e g Unit A drifted away from the noise cluster as the experiment proceeded To display the Clusters vs Time View Select the View Clusters vs Time View menu command or press the E
422. ype 14 Neuroshare Native nsn 15 RC Electronics 16 Axion 17 generic binary 18 generic Neuroshare 19 reserved 20 multiple files OMF ImportParameters specifies a filename for import parameters ofi file to be used for binary file import FilterFreq sets the low cut filtering frequency in Hz to use with ForEachChannel Filter FilterType set the type of the filter to apply can be Butterworth Bessel or Elliptic FilterPoles sets the number of poles to use for the filter can be 2 4 6 8 10 or 12 UpSampleType sets the type of interpolation to do for upsampling None 1 Linear 2 Spline UpSampleF actor sets the interpolation factor for upsampling e g 2 means X interpolation UpSampleMinFreq UpSampleMaxFregq can be used to select which continuous channels will be upsampled e g if a file contains some continuous channels sampled at 1000 Hz and others sampled at 4000 Hz setting a UpSampleMinFreq of 30000 will ensure that only the 40000 Hz channels are upsampled DetectNPW sets the number of points per waveform to use for ForEachChannel Detect DetectNPre sets the number of pre threshold samples to use for ForEachChannel Detect DetectSigmas sets the detection threshold as a number of sigmas away from the mean of the Peak Height Histogram the value can be negative for negative thresholds DetectMicrovolts sets the detection threshold
423. ze Icy Gray Text Below Toolbar Icons Hide Window Title Bars Theme Group The top group of items are theme toggle items that may be selected to apply to the user interface look and feel Only one of the themes may be selected at a time e Text Below Toolbar Icons This item is a toggle item to show or not show text below the toolbar icons e Hide Window Title Bars This item is a toggle item to hide or show win dow title bars e Layout Clicking on Layout displays a menu as shown below A layout is the size and placement of the windows on the screen It also remembers the number and placement of toolbars Version 4 0 A 11 Appendix A Navigating the Plexon Ul Reset to Default Layout Apply Layout 1 Apply Layout 2 Load Layout From File Save As Layout 1 Save As Layout 2 Save Layout to File e Reset to Default Layout Clicking on this item resets the layout to the factory default e Load Layout Group Clicking on Apply Layout 1 or Apply Layout 2 applies one of the standard layouts to the user interface Clicking on Load Layout From File will allow you to select a layout file to apply to the user interface e Save Layout Group Clicking on Save As Layout 1 or Save As Layout 2 saves the current screen layout as one of the two standard layouts You may also click Save Layout to File to save the current screen layout to a file that can be loaded by the Load Layout From File item

User Guide - Plexon Inc

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