Additional file 2
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1. 0216 160645955910046 003 2 0 1L0470619534053446 002 ta SZlOOlLZZ04 950 606 00Z LeJO0 1550042003 15526e 001 The data organized in this way can be found here http www spikesorting com simulated data aspx They are the same simulation datasets used by R Quian Quiroga Z Nadasdy and Y Ben Shaul Unsupervised Spike Detection and Sorting with Wavelets and Superparamagnetic Clustering Neural Computation 16 1661 1687 2004 f Continuous FE this is the format that uses FSPS software for continuous data acquisition HDD streaming button The file has 8032 bytes 1004 164 values header information First four values in the header are 1 The number of channels depends on how many input physical channels are selected in the I channel control 2 The number of samples in one data chunk for each channel 1000 is a default 3 The digitalization rate as samples s 10000 is a default 4 The number of reserved U8 lines the default is 0 At the moment the rest 1000 64 values reserved for multi electrode array and brain computer interface in progress UNIFE Section of Human Physiology 2012 FSPS User Manual Page 15 of 35 After the header comes a series of chunks A chunk consists of four parts length integer 32 eight U8 values can be 0 or 1 for digital events af any 32 integer of millisecond timer value that has been split into its component bytes four U8 values an2. International settings To fix this please set English language for non Unicode programs and or apply English to the your day amp time system format b Run the programs as an Administrator 1 e check Administrator box in the Privilege level menu see Compatibility Tab of the FSPS exe property c Try to select another more direct Programme folder during installation procedure C PSPS for example Q FSPS software starts but do not perform classification when I press TEST button Thus the only one class blue spike shapes is visualized A This may happen on some computers running 64 bit version of Windows To make the classifier working correctly you should run FSPS software in the WindowsXP compatibility mode see Compatibility Tab of the FSPS exe property However we are working to make it compatible with all 64 bits operating systems UNIFE Section of Human Physiology 2012 FSPS User Manual Page 35 of 35 Glossary FCM Fuzzy C Mean see Tutorial for details http home dei polimi it matteucc Clustering tutorial html cmeans html FSPS _ An abbreviation from Fuzzy Spike Sorting see www spikesorting com HT Italian Institute of Technology http www iit it PCA Principal Component Analysis see here http en wikipedia org wiki Principal component analysis for details PCs Principal Components PSVD Partial Singular Value Decomposition see The Compu
3. RSE l Preview Terminal configuration Channel for Min Samplesich mima Minimal Input 52 10 00 100000 Frite l Sample mode Max Sample Rate Hz Maximal Input 12 00 10000 Digitalization rate Hz Number of samples to be acquired during test the same parameter will be applied acquisition for example 100 000 samples will for Analog Output channel take 10s of recording time Press button to view the signal at the selected channel 1n separate window Fig 1b Figure 1b Samples to Read To Devil MO so UNIFE Section of Human Physiology 2012 FSPS User Manual Without NI Digital Acquisition Board Page 9 of 35 If you don t have an NI DAQ connected or wish to use FSPS software solely to analyse existing recordings offline you should switch OFF the control button default value right after running the program Fig 1c Thus further operations and analysis will be performed on existing recordings selected in the tab Figure Ic MAIN Filtering Spline save Source DAQ DAQ DAQ control ER Cc Preview Physical Channel Terminal config alan pies TR 4 Dev1 ail gt RSE Terminal configuration ee Un SYNA Min Samples Ch Sample mode Minimal Input 10 00 100000 Finite gt Sample mode Max Sample Rate Hz Maximal Input 5 10 00 10000 Digitalization rate Hz the same parameter will be applied for an Analog Output channel Number o
4. additional information could help the experimenter to control task execution or to align recording neuronal activity to one of these external events The current version of the software supports up to different 6 TTL channels thus 6 events however their number depends mainly on the type of DAQ board utilized One example of their application in an example experimental task is shown below The task in question was the execution of goal directed movement by the left hand of the monkey during recording from premotor cortex area F5 of the right cerebral hemisphere Raw signal gt Spikes hardware _ threshold discriminator 1 2345 6 2 TTL events during task execution The duration of the trial was 3s In this time the monkey reached out his hand to the handle of the small door of the box opened it and took the food as a reward The Figure shows as well as the neuronal activity recorded by the electrode the external events characterizing this behavioural task 1 green line the hand of the monkey is still in its initial position 2 pink the hand crosses the infra red barrier in front of the door 3 upper yellow touching the upper sensor of the handle 4 lower yellow touching the lower sensor of the handle 5 red main trigger contact with both the upper and lower sensors of the handle 6 blue taking the food from the box Trigger interna
5. is recommended Hard Disk drivers free 500 Mb of free space LYNN NNN 3 Software v Windows XP 2000 Vista Vista x64 7 7 x64 NI DAQmx drivers free at www ni com v LabVIEW Run Time Engine 2009 free at www ni com for new data acquisitions or online classification only some netbooks running Windows XP or Windows 7 are also supported UNIFE Section of Human Physiology 2012 FSPS User Manual Page 7 of 35 Installation The installation process is performed as follows LabVIEW Run Time Engine 2009 Windows 2000 7 7 x64 Vista Vista x64 XP 32 bit Standard RTE free at www ni com 160 MB NI DAQmx Run Time Engine 9 3 Core Windows 7 64 bit 7 x86 Server 2003 R2 2 32 bit XP x86 Vista x64 Vista x86 Server 2008 R2 64 bit free at www ni com 3 Full version 1 First install LabVIEW Run Time Engine 2009 Windows 2000 7 7 x64 Vista Vista x64 XP 32 bit Standard RTE Installation file LVRTE90Ostd exe 160 MB provided to registered users free of charge by NI www ni com Alternatively it can be downloaded from http www spikesorting com Download page 2 Next install DAQmx device drivers supplied together with your NI DAQ device check at www ni com for details If you don t have NI DAQ drivers installed even if you intend to use FSPS software exclusively for off line analysis without acquisition board you should nevertheless proceed with installati
6. nit Mode Random E gt ee 40 0001000 Initial parameters of FCM clustering algorithm Init Lower Bound 0 00 Init Upper Bound 1 00 Use Random Seed v is240cc700r Init Random Seed w 1234567890 Norm FCM init FCM Status Performance Performance Tab contents Read data ma Information about the absolute time of each off line running SPK extraction 6926 procedure in milliseconds Mean subtraction 331 SVD 216 9222 classes FCM 1 17219 CM overall lt 5997 clustering viz 1 Bo Labeling 837 UNIFE Section of Human Physiology 2012 FSPS User Manual Page 16 of 35 Chapter 2 4 Control Tab 3 Visualization and Statistics Shapes Tab contents Sm sa a 4 Shapes STH Rasters 30 Quality Measures Indices About dao O E an o M lassi med Change a with Class3 f clusters a b C d The number of spikes shown for each cluster can be adjusted using the tool SPK show found on control window 6 see Figure 2 the default is 50 The white spike profile is an average of that class button a auto scales X and Y axes the first window shapes marked in blue in our specific case button b performs the same operation as a but takes into account the highest amplitude found in any of the classes shown Clicking button c applies the scales and ranges selected for the first window to windows 2 12 You can also adjust Y sca
7. slightly improve overall classification performance on some slow computers ON is default value 4 control button to switch ON and OFF sound generation and reproduction both clicks or original signal see 6 through line out of the computer board OFF is default value NO 5 Actual threshold level for spike detection algorithm 6 Original Classified control button consents reproduction of Original signal picked up by electrode or Clicks pertaining to classified waveforms though line out of computer sound board Classified is default value 7 Signal Inversion switches the inversion of the input signal ON and OFF 8 HDD streaming switches ON and OFF data storage to HDD of the computer with a filename dat indicated above as a Flow Chart indicating single unit activity 9 Pull down menu for selection of the isolated unit to be acoustically reproduced and visualized in 12 10 Information panel displaying number of Classes and Features used 11 STOP control button stops on line monitoring and closes current window UNIFE Section of Human Physiology 2012 FSPS User Manual Page 30 of 35 12 13 14 15 Graph indicating spike shapes that successfully passed classification procedure and satisfied the parameters of class selected in 9 Graph indicating all spike shapes extracted at current time instant Moving cha
8. under the direct supervision of an experienced researcher or licensed practitioner 4 This software should not be utilized without careful review of the complete instruction manual provided in the application folder or available at the web address http www spikesorting com documentation aspx 5 Periodic updates for the software may become available and the user accepts their obligation to register any changes in their e mail address at www spikesorting com if registered should they wish to be informed about new releases Users are also urged to check the www spikesorting com website for announcements of software upgrades 6 LIMITATION OF LIABILITY TO THE FULL EXTENT PERMITTED BY APPLICABLE LAW THE LICENSEE RECOGNIZES AND ACCEPTS THAT A THE SOFTWARE IS SUPPLIED AS IS AND THE AUTHOR DOES NOT GUARANTEE THAT THE SOFTWARE IS FREE OF BUGS FLAWS OR DEFECTS B THE AUTHOR DOES NOT GUARANTEE THAT THE SOFTWARE MEETS THE SPECIFIC NEEDS OF THE USER AND C THE AUTHOR EXPRESSLY DISCLAIMS ANY RESPONSIBILITY FOR ANY UNIFE Section of Human Physiology 2012 FSPS User Manual Page 5 of 35 DAMAGE LOSS OF PROFITS LOSS OF BUSINESS LOSS OF DATA DAMAGE TO COMPUTERS OR PERIPHERAL EQUIPMENT OR ANY OTHER DIRECT INDIRECT INCIDENTAL CONSEQUENTIAL SPECIAL OR PUNITIVE DAMAGE OR ANY LOSSES OF ANY KIND ARISING DIRECTLY OR INDIRECTLY FROM THE USE OF THE SOFTWARE CAUSED TO THE USER PATIENT OR ANY THIRD PARTY THE FOREGOING LIMITATIONS A
9. A FSPS software is available at http www spikesorting com UNIFE Section of Human Physiology 2012 FSPS User Manual Page 4 of 35 Terms amp Conditions Agreement FSPS is a computer programme for the fast and accurate automatic isolation of neurons during electrophysiological recordings it can be used for signal acquisition visualization classification and analysis of neuronal spikes For more information regarding the current version of FSPS please e mail info spikesorting com Prior to download and use the user acknowledges that 1 FSPS is distributed under Creative Commons Public License CCPL BY NC ND and can be used for academic applications providing they properly reference our work in any publication that uses results generated by FSPS software Under this CCPL you are free to Share to copy distribute and transmit the work Permissions beyond the scope of this public license are available at www spikesorting com Under the following conditions You must attribute this work referring to the authors Oliynyk A Bonifazzi C Montani F and Fadiga L and citing the source URL You may not use this work for commercial purposes You may not alter transform or build upon this work OOO 2 NO signal acquisition features or on line classification functionality of FSPS are limited under CCPL BY NC ND 3 The software is a biomedical tool that should only be used by or
10. General Information A challenging issue in computational neurosciences is the development of algorithms to detect and sort the spiking activity of individual neurons from extracellular recordings While many algorithms for spike sorting exist the problem of accurate and fast online sorting of spikes still remains to be solved In answer to this problem this novel software tool FSPS Fuzzy SPike Sorting has been designed to optimize fast and accurate detection off line sorting and on line classification of neuronal spikes with little or no human intervention The FSPS algorithm is based on a combination of the following main methods a b c d e f g SPLINE interpolation for high resolution alignment of extracted spike waveforms Singular Value Decomposition SVD and its modification Partial Singular Decomposition PSVD for fast and highly accurate online pre processing of spike shapes Scree Test Optimal Coordinates SOC for optimal selection of the number of features to retain Automatic identification the number of clusters using a histogram based method of dataset segmentation Fuzzy C mean FCM for the clustering classification of PCs in the multi dimensional space feature Mahalanobis distance measurements Quantitative quality assessment of the resulting clusters irrespective of their size The software was entirely written and compiled using LabVIEW 2009 National Instruments US
11. PPLY EVEN IF THE SOFTWARE CEASES TO FULFIL ITS ORIGINAL AND INTENDED PURPOSE 7 The author does not guarantee the user any form of training support or instruction regarding use of the software By installing FSPS you agree to the terms and conditions listed above Author Dr Andriy Oliynyk Section of Human Physiology Department of Biomedical Science and Advanced Therapy University of Ferrara Via Fossato di Mortara 17 19 44121 Ferrara FE Italy TO OBTAIN A COMMERCIAL LICENSE PLEASE DELETE CCPL FILE LICENSE PNG MANUALLY RUN THE SOFTWARE AND PROVIDE US WITH AN AUTHORIZATION CODE NECESSARY FOR YOUR INDIVIDUAL COMMERCIAL LICENSE PLEASE ALLOW UP TO 48 HOURS FOR AN ACTIVATION FILE TO BE DELIVERED USB SECURITY KEY WILL BE RELEASED AND DELIVERED AFTER FSPS COMMERCIAL LICENSE PURCHASE UNIFE Section of Human Physiology 2012 FSPS User Manual Page 6 of 35 Technical Requirements Minimum hardware software and operating system specifications Hardware 1 National Instruments I O Digital Acquisition Board having at least v 1 Analogue input channel sample rate 10 kS S with a Measurement Type Voltage 2 Computer Processor form factor Pentium II or above RAM 1 GB or more Display at least 1280 x 800 pixel resolution 1280 x 1024 1s recommended Video Card 128 Mb 24 bit colours resolution 1024 x 800 or higher Monitor 15 inch resolution 1024 x 800 or higher 1280 x 1024 mode
12. User Manual English Version 3 0 0 4 Date 30 06 2012 Prepared by 2 3 0 10 University of Ferrara Faculty of Medicine Andriy Oliynyk 30 06 2012 Department of Biomedical Science and Advanced Therapy Section of Human Physiology Revision History 2 0 0 32 31 10 2011 Update 6 2 1 0 84 24 01 2012 Update 7 2 2 0 15 01 02 2012 Update 8 FTDI support is included Update 9 This version introduced a number of significant enhancements including improved classification performance suitable now for multi electrodes 2 3 0 10 10 06 2012 rewriting the core code without commercial third part add on improvements in visualization during continuous recordings cluster information added export data into txt file export cluster center information some bug fixed and more The implementation of Creative Commons Public License CCPL BY NC ND for 3 0 0 4 30 06 2012 academic non commercial use current version FSPS User Manual Page 2 of 35 Table of Contents Revisto IIS COTY ios se nessa a Sia aa pees sce eaten DS e l TOLO OS e a E E EA E E E A E 2 enera OTO pester e E E A A E E EEIE 3 Terms Ce Conditions Fy Sie Ce OL serieen ge r RR EEEE 4 Tonn eR CS cosas A A A A E 6 nte AAC 6 APR RR REDOR OR EE ETEA E E O 7 Chapter 1 Getting Stard sesa a R E E R SE AT a 8 VEND a OA e E E E E 8 NV ALO WGA TOS IAI BOATO a usasse stop as shen cers dos Tapio E pi eia id sa 9 Chapter 2 Continuous Signal Acquisition and Of
13. alogue input signal It must be the same as that used during the TEST clustering procedure This parameter is set up automatically but can be manually adjusted here if required not recommended sample ch the number of samples to be acquired during each measurement session For instance at a digitalization rate of 10 000 samples s the acquisition of 500 samples will take 20 ms default value This can be adjusted to your specifications Chapter 4 2 Flow Chart Firing rate Tab Firing rate STAT AciprogrammiiFsPs _recordings O dat i tt w 3 sa spikes per min oe i 11 27 46 11 27 58 30 03 30 03 This Flow Chart shows the firing rate of isolated single units At the top part of this Tab there is an indicator showing the file name and the path for data storage when the button is ON Pressing the button top right creates a new empty file The filenames will feature progressively increasing numbers 1 dat 2 dat 3 dat etc UNIFE Section of Human Physiology 2012 FSPS User Manual Page 32 of 35 STAT Tab Firingrate STAT Base path 9 C Pragrammi FSPS data y dat Iteration Duration 1 Spikes found 16 Spikes cancelled fp 0 Classification performance Latency ms Number of Steps 422 SPLINE 0 Training Error 0 00002994 MEAN O Test Error D 00000000 svD O Partition Coefficient 95039369 Fem 27 P
14. array of single precision SGL elements of measured data multiple of a number of samples in one data chunk for each channel see above if you need just export the data recorded with FSPS software into your software you may use Export raw data at the Tab It saves your current channel in the txt file without the need to build your own file converter there are two additional buttons that allow a specific part of the binary file designed primarily for long GE files to be read allowing it to be processed in batches The number of such batches is counted automatically when this option is selected For triggered FE data the same buttons allow solely a specific trial to be read A Note that for batch processing of long files it is necessary to switch OFF all automatic options at the Main Tab to maintain the same parameters for all parts of the file The report if you want generate and save it using Save Tab will show information from these batches as one continuous file Chapter 2 3 Control Tab 2 FCM status and performance information Norm FCM init FCM Status Performance 600 Tab contents 400 A histogram showing the distribution of norm values for every score 300 of PCs The histogram is automatically thresholded red horizontal line to eliminate the noise content RE RE UU So esl E e O 5 10 1520 25 30 35 40 45 50 55 Norm FCM init gt FCM Status Performance son FCM init Tab contents
15. atic reduction of the amplitude of an action potential when the cell fires in a sustained high frequency burst systematic slow drift of electrode etc UNIFE Section of Human Physiology 2012 FSPS User Manual Page 22 of 35 A bout your license info neea wre a posto Essa CREED FUZZ Spike Sorting Software Please refer to this page to check your License The DataMatrix image confirms the presence of the License file while the green indicator below shows its type and validity Note that the Creative Commons Public License CCPL BY NC ND for academic non commercial use is included in the installation package UNIFE Section of Human Physiology 2012 FSPS User Manual Page 23 of 35 Chapter 3 Triggered Acquisition and Off line Spike Sorting Figure 3 Continuous acquisition Triggered acquisition O O O O O RO TT aT a TT TT i Ta TT TTT TT TTT TTT TTT p BC NO OT TT O TTT ATT TT TT TT TTT TTT TTT TT 10 pC TT TT Tt tT e TT TT TT TTT TTT TTT ToT TT TT HLSd suang eb peer na ayes a Ea pam Es Ea ER e Grasping the food with left hand Ja66u L 5 a Folder path 6 CEspsisetuplexamplesiFE 12130 24 7 E amplitude a 7 3 04 mm 1 ke a e 1 1 7 1 1 i ea eon 0 5000 10000 15000 20000 25000 The same as for Continuous Acquisition THR WAVE PRT RECALL S Condition total Record N Scrolling Recor
16. aved depending using the Original Classified Figure 4 switch Original saves raw signal data while Classified saves timestamps for isolated spikes only UNIFE Section of Human Physiology 2012 FSPS User Manual Page 33 of 35 Examples and Supporting Files Example files are located in the installation folder FE contains triggered acquisition from the Monkey Premotor Cortex area F5c during goal directed grasping movement The acquisition is triggered at 1 second when the monkey touches the sensor Experiments were performed at the University of Ferrara GE this folder contains recordings of cortical neuronal discharges acquired by nanotubes at IIT Genova The signal is unfiltered and activation of software filtering option is therefore necessary Due to long recording you may need to process it in batches UD several human recordings from internal GPi during Deep Brain Stimulation Udine QUI a text file usually as data txt contains simulation data originally proposed and published by Q Quiroga et al 2004 doi 10 1162 089976604774201631 More recording to test the SPS algorithm can be downloaded here http www spikesorting com examples demo aspx UNIFE Section of Human Physiology 2012 FSPS User Manual Page 34 of 35 FAQ amp Troubleshooting Q FSPS software does not run after installation Why A a This may happen on some computers running Windows 7 with particular
17. d 18 Continuos 151617 UNIFE Section of Human Physiology 2012 FSPS User Manual Page 24 of 35 Legend 1 External Tabs Tab control 0 switch between and Triggered acquisition Raw signal graph trial 1 of 12 ON Spike location spikes are determined by hardware voltage discriminator external to the FSPS programme Coloured lines correspond to different external events during task execution Name of the destination folder Update graphs can be used to redraw raw signal graphs 2 when necessary allows adjustment of Y scaling factor for raw signal graphs p 10 in pull down menu N OOA Oo Group labelling information for each triggered acquisition set 9 Tab Control 1 advanced triggering settings and PSTH based on spike occurrence determined by hardware voltage discriminator 10 Group graphs and indicators representing a particular recording session 12 trials in this case 11 Group control to include trials for PSTH composition 12 Fast selection of all trials to be included in hardware based PSTH graph 13 Fast deselection button for hardware based PSTH graph 14 Fast automatic extraction and visualization of PCs scores for each trial 15 control enables triggered acquisitions and recording functions 16 19 16 control allows automatic adjustment of the Record n and Condition 1f many when the acquisition of the current test is complete 17 control
18. dex Lratio IS superior and so is the default quality measure In the vast majority of cases values below 5 threshold means acceptable cluster quality Values greater than 5 are usually associated with poor clusters with reference to their compactness and distance from other clusters in the dataset Exact Lratio values for each cluster are shown in the graph legend For more details see doi 10 1016 j neuroscience 2004 09 066 UNIFE Section of Human Physiology 2012 FSPS User Manual Page 21 of 35 Indices Tab contents lt ioms gt 10ms Burstindex lt SOms gt 50ms PauseIndex Mean firing rate Color a aon 5900 ms 00 1200 zs m Find ao 70400 001 17900 s os agro w saw o of sao 029 gas o o 4i00 sow onm 10 25400 062 na mea DSG 93 00 1147 00 Feature 1 Feature2 Featue3 Burst Index BI defined as the number of ISI lt 10 ms divided by the number of ISI gt 10 ms Pause index PI defined as the number of ISI gt 50 ms divided by the number of ISI lt 50 ms For more details see doi 10 1016 S0090 3019 99 00030 0 The table below shows the coordinates of cluster centers The button is experimental It allows tracking of clusters in the feature space when parameters of spike waveform is changing due to some deterministic non noise influences e g small periodic displacements of the location of electrical activity relative to the microelectrode system
19. f line Spike Sorting seeeesssseeeeeeeeeeeees 10 Chapter 2 1 Control Tab O Continuous or Triggered ACQUISITION ccccccceeeeeeeeeeeees 11 Chapter 2 2 Control Tab 1 Basic I O Parameters Control Tab ee ccececcecceeeeeeeees 12 Chapter 2 3 Control Tab 2 FCM Status and Performance Information 15 Chapter 2 4 Control Tab 3 Visualization and Statistics cccccccccccccccssssesseeeeeeeeeeeeeeeees 16 Chapter 3 Triggered Acquisition and Off line Spike Sorting cecccccccccccecessseeeeeeeeeeeaeeeees 29 Chapter 3 1 Control Tab O Continuous or Triggered ACQUISITION ccccccccceeeeeeeeeeees 25 Chapter 3 2 Control Tab 1 Hardware Info amp Settings i eecccccccccceesseeeeeeeceeeeeeeeeeeees 25 Ae CC NC SOL e sacra eaioniadac corevsaian tunes aaisanis 2 Chapter 3 4 Get Instant Principal Components cccccccsssseeeeeceeceeeeaeeseeeeeeeeeeeeeeeeees 28 Tate d On ine SPU OTN ioen E E T E E SE 29 Chapter 4 1 Input Output Paramictens cccarieessiecssaacencteetsiasasaietarieessiovasenceucteetetasasasatarieeseticds 31 Sete de A A E a A E E A EE AA T 31 Chapter 4 3 HDD Streaming ssoeoeeessssssssseerrsssssseserrrssssssssererssssssssereresssssseeeeressssseeeerees I IX ain Fes AC aa aa PE da SE E E 33 FAQ amp OUI CS OO bi SOR OO O DN O 34 OES eM RR GARD RR AP DD O AR DR 35 UNIFE Section of Human Physiology 2012 FSPS User Manual Page 3 of 35
20. f samples to be acquired during test acquisition for example 100 000 samples will take 10s of recording time UNIFE Section of Human Physiology 2012 FSPS User Manual Page 10 of 35 Chapter 2 Continuous Signal Acquisition and Off line Spike Sorting Figure 2 ape RE 18 ad a dt dd DD ll UIII IU AL alba AIL aah i Samples time 1 1 i 1 i 1 1 i 1 i i i i i i i 0 0 0 2 0 4 0 6 0 8 1 0 1 2 1 4 1 6 1 5 2 0 2 2 6 2 8 3 0 3 2 3 4 3 6 3 8 4 0 ihe 4 4 4 6 4 8 5 0 1 4 3 J J j2 2 4 Shapes PSTH Rasters 3D Quality Measures Indices About DAQ MAIN Filtering Spline Save Source DETECTION SD value az 1 10 Exponent 5 THRESHOLD clusters New E Aut FCM prototype features 3 6 5 19 5 05 0 35 wth cassa A C00 clusters 8 9 10 11 1213 14 15 16 Control Tab 0 switches between Continuous and Triggered Acquisition Control switch for X axis scale samples or absolute time is used in graphs and PSTH Raw signal graph Spike locator the colour of the dot corresponds to the colour of the spike shape in 13 Control Tab 1 selection of input parameters see Chapter 2 1 for details O E OUN Group automation indicators Signal THR the voltage threshold level for the spike detection algorithm Batch No the number of the data chunk that is currently being processed SPK found the total number of spikes detected in the data set being processed Hi
21. in crisp clustering 1 1 is the default value o DAQ MAIN Filtering Spline Save Source Filtering Tab options On Off is activating deactivating software bandpass Butterworth filter The default is Off Filter parameters high cutoff freq Hz is the high cut off frequency in Hz high cutoff freq Hz Jt must be greater than low cut off frequency and fulfil the 7000 Nyquist criterion low cutoff freq Hz Filter C of rder a 400 low cutoff freq Hz is the low cut off frequency in Hz and must fulfil the Nyquist criterion If low cut off frequency is less than or equal to O or greater than half the sampling frequency value the algorithm is not working The low cut off frequency must be less than the high cut off frequency order specifies the filter order and must be greater than 0 The default is 5 If the order is less than or equal to O the algorithm signals an error UNIFE Section of Human Physiology 2012 FSPS User Manual Page 13 of 35 DAG eos t See e Solin Save Source MAIN Filtering Spline Tab options INTERPOLATION MODE can be 0 1 2 3 or 4 The default is 1 It determines slightly different strategies of waveform extractions and their alignment depending on spike duration acquisition rate etc Detailed information is indicated in the table below INTERPOLATION Inverse signal FF Length of Location Inte
22. l tab hardware settings Before starting acquisition use this Tab to define desired digitalization settings and trigger parameters UNIFE Section of Human Physiology 2012 FSPS User Manual Page 27 of 35 1 gt 2 gt rising o i Bs 3 fioo rios E 4 5 1 The number of pre triggered samples In this specific case 10000 samples equal 1 s Digitalization rate specified in 3 is 10000 samples s 2 Total duration of the trial In this case 30000 equals 3 s Digitalization rate specified in 3 is 10000 samples s 3 Digitalization rate specified samples s 4 Physical channels for event TTL signals 5 Direction of electrical edge for trigger 6 Adjustments for TTL level 7 Physical trigger channel Chapter 3 3 Create New Set This interface window emerges after pressing the button 19 in Figure 3 It allows you to initiate a new Recording set composed of the desired number of experimental Conditions Each experimental Condition contains 12 or fewer signal acquisitions or trials You may choose destination folder where your data folders will be created after pressing the button Create Please remember to give meaningful names for Conditions since they are visualized during recordings UNIFE Section of Human Physiology 2012 FSPS User Manual Page 28 of 35 Chapter 3 4 Get instant Principal Components This interface window emerges after pressing the bu
23. le parameters manually by entering its numerical values in the first window click button c to apply it to all of the other windows Command group d allows you to change the order of the classes manually by clicking on button Change or merge one class with another by clicking on the button Merge clusters To return to the initial cluster and order switch OFF green LED located to the right of the switch UNIFE Section of Human Physiology 2012 FSPS User Manual Page 17 of 35 Adina PSTH Tab contents T PSTH TH Biter 3D Quality Measures Indices About ie Autoscale o es a ap Pino 4 E iso PSTH histograms are particularly useful in triggered trials but can also be used in continuous acquisitions Histogram length and height can be set up automatically when a and e controls are ON default settings or manually controls a and e Use Switch 2 see Figure 2 to change X scale between samples or seconds Both X and Y scales can be set manually To change the duration of the bin select the appropriate bin parameter control b button c extends the first graph s settings to the other graphs displayed as described above for the Tab Detailed information about the contents of each bin for each histogram can be obtained by pressing the button d This exports the information to Excel tables one for each class Note Micros
24. oft Excel must be installed for this to function UNIFE Section of Human Physiology 2012 FSPS User Manual Page 18 of 35 contents Pe tel lo ma Shapes PSTEI Rasters o Quality Measures Indices About Displays Raster plots for each detected class and trail Button b switches ON and OFF vertical grid Grid colour can be chosen by clicking on a button c function is the same as described above for the Tab UNIFE Section of Human Physiology 2012 FSPS User Manual Page 19 of 35 Tab contents Shows the 3D Scatter Plot of the first three PCs accumulating maximal variance Other PCs can be viewed by selecting their numbers using the controls in the top right corner Other graph properties 1 Click and drag the graph to change the view direction 2 Press the lt Shift gt key when you drag the mouse up to zoom in and down to zoom out 3 Press lt Ctrl gt key when you drag the mouse to move the graph around 4 Change the view direction mode using the projection palette to the lower right of graph UNIFE Section of Human Physiology 2012 FSPS User Manual Page 20 of 35 Quality Measures Tab contents Any kind of clustering or classification needs an objective measure of its quality the most conventional indices associated with FCM including partition coefficient partition entropy and proportion exponent are incorporated in the Toolbox although our tests show that the in
25. on of NI DAQmx Run Time Engine 9 3 Core Installation file NIDAQ930f2Core zip 155 MB is provided to registered users free of charge by NI www ni com Alternatively it can be downloaded from http www spikesorting com Download page 3 Install FSPS program http www spikesorting com approx 50 MB Download page Uninstall notes Use Uninstall FSPS file unins000 exe located in the FSPS application folder to remove the software from computer To uninstall LabVIEW Run Time Engine 2009 and NI Device Drivers you should use the standard tool on the Windows Control Panel UNIFE Section of Human Physiology 2012 FSPS User Manual Page 8 of 35 Chapter 1 Getting Started With NI Digital Acquisition Board NI DA To use FSPS with a NI DAQ connect the latter to the computer and configure your NI DAQ device as specified in the relevant manual before running the FSPS programme Next run the programme fsps exe from the FSPS destination application folder C Program Files fsps exe by default As soon as the programme starts configure your NI DAQ board To do this activate the control button at the tab Control Tab 1 and set up I O parameters against your particular board configuration Fig la Figure la MAIN Filtering Spline Save SOUICE DAG DAD DAQ control gt _ gt Physical Channel Terminal config Analog Input gt pev1 aio
26. performs the same operation described below in 18 automatically O 99 099 incrementing Record n and starting a new trial It deactivates when Record n reaches a value of 12 and all your Conditions are complete 18 prepares to record trigger event by default at PFIO of NI DAQ upon which it writes overwrites acquired signal at the position indicated in control 22 19 allows the creation and design of your acquisition set by selecting and naming different experimental Conditions 20 Indicates the current Condition in the Set 21 Control button PLA Y converts the raw signal of the current trial indicated at Record n control into sound file WAV format and reproducing it through computer sound board 22 control serves to navigate between trials write overwrite or convert into WAV format To select particular trial to overwrite it for instance the Scrolling button must be switched Off 23 Indicates the total number of Conditions in the recording set ok 24 Indicator of the current Condition UNIFE Section of Human Physiology 2012 FSPS User Manual Page 25 of 35 Chapter 3 1 Control Tab 0 Continuous or Triggered acquisition Active _ Allows you to choose between Continuous acquisition Triggered acquisition continuous Chapter 2 and this chapter acquisition depending on your needs and experimental design Wii T T TTT T Chapte
27. ph p 3 at the Figure 2 in separate txt file Tab options Folder path use dialog is a path control that allows you to select a file folder with data Before selecting the folder you have to select the file type from drop down menu At the moment there are 6 different formats available UNIFE Section of Human Physiology 2012 FSPS User Manual Page 14 of 35 a Binary GE a stream of unsigned 16 bit integers U16 having little endian byte order There is no header information DAQ b Text UD TXT for text files as follows ME we Source MAIN Filtering Spline T see X Y YY Folder path control where X 1s a progressive number of samples gC IFSPS examples FE 12130 to delimit fields in the text file Y YY 1s a value in which the dot is used as the decimal Read DATA ee all at once ra separator see example below small files ils C m Scroll E alitomatically File Type Continuous FE 1 5 20 2 7 40 3 6 97 4 4 57 5 1 67 6 1 05 etc c Triggered FE reads data from the 16 bit Integer file which contains 12 rows corresponding to the triggered acquisition trials d Binary MAP reads map files generated by the AlphaMap software from Alpha Omega Nazareth Israel This converter 1s experimental e Simulated Qui another kind of TEXT file to import data saved in engineering format as follows Ea 00 242497 LI LE DU Z Os 124 LOCO 214060906 D0UZ A
28. r 3 2 Control Tab 1 hardware info amp settings PSTH internal tab hardware based 25 gt q 20 ESS 10 ir i 0 1 1 1 i i 1 0 5000 10000 15000 20000 25000 30000 gt Commonly electrophysiological recording systems contain an option to perform spike isolation using hardware voltage discriminator This makes it possible to roughly characterize neuronal activity and its specificity instantly and during recording process This function is also offered by this software in parallel with the Fuzzy Spike Sorting procedure Please note that to acquire the signal from your window discriminator hardware in our case it is channel 2 you need to reserve a separate I O channel at your DAQ A can be used to switch auto scale for PSTH graph ON green or OFF dark UNIFE Section of Human Physiology 2012 FSPS User Manual Page 26 of 35 Events internal tab supplemental TTLs The average duration MEAN SEM Events of each external event occurring during A Base Piate E Sottom Trigger Upper _ Teod execution of the task is given at this Mean s 2 0026 0 8627 0 2581 0 2537 0 3235 0 2263 E SEM s 0 3546 0 0828 0 0134 0 0135 0 0220 0 0559 Tab Indeed during your electrophysiological study single unit recordings or whatever you may need to control some other external parameters such as the duration of button or sensor holding precise touch timing and or kind of kinematic data etc This
29. roportion Exponent 1 00000000 Waveforms O Sound 0 Classification Entropy 0 08233612 Trained Labelled The Tab shows some additional information about the on line classification module performance Base path shows the path for the V dat file with the cluster prototype for your current recording It is updated each time when you press the TEST button see Chapter 2 Figure 2 Spikes found shows the number of spikes detected in the current time frame and subjected to the automatic classification algorithm Spikes cancelled shows the number of spikes in the current time frame that have been cancelled by our false positive removing algorithm Classification performance group indicators showing basic Fuzzy C Mean parameters and quality measurements Latency ms is a duration of each critical step of spikes classification in milliseconds Chapter 4 3 HDD streaming While monitoring neuronal activity during experiments or clinical tests you may need to record some important fragments for future in depth analysis Pressing the button HDD streaming will start this process saving the data in indicated file To start a new file click the button Path to data storage folder Create New dat file Firing rate stat 3 C Programmi FSPS _recordings 0 dat Bu 70 The number of your dat file will automatically increase You can select the source of your data to be s
30. rpolation Overall length Cut out Length of extracted rate times of interpolated samples resulting waveform waveform beginning end waveform samples samples samples Inverse signal inverts the signal ON OFF unchanged is default DAQ Save Tab options Save clustering results control button allows the results of clustering classification to be saved automatically when ON At the end of each run of MAIN Fitering Spline Save Source Save clustering results sa clustering classification algorithm by pressing the TEST Export raw data button it saves a text file tcs in the same folder The C D file contains OFF 1 Peak occurrence time 1 value in each row 2 Class label Oo value in each row 3 3 the end of each row is an interpolated waveform of the spike Therefore the number of rows corresponds to a number of extracted spikes while the number of columns depends on the length of the waveform consult the table above control button allows Principal Components to be saved automatically when ON At the end of each run of clustering classification algorithm by pressing the button it saves a text file psvd in the same folder The file contains 1 First column Class label rd value in each row 2 Second column till the last one Principal components like PC1 PC2 PC3 PCk control button allows to save your current channel visualized in Raw signal gra
31. rt indicator displaying raw signal Flow chart displaying current firing rate of isolated and classified single units Note settings are determined during TEST procedure offline and transferred from interface window shown in Figure 2 UNIFE Section of Human Physiology 2012 FSPS User Manual Page 31 of 35 Chapter 4 1 Input Output parameters I O Parameters I Channel The name and the number of DAQ Analogue Input channel connected to recording electrode Its activity is I Channel O ID E 7 a l L Devi fail Mo visualized in the Moving Chart indicator see Figure 4 P7 9 J default it 1s the same channel that has been used in TEST Max Rate clustering procedure Fig 1a This parameter is set up automatically i 10 00 7 i 10000 but can be manually adjusted here if required Min samples ch J 10 00 500 Max maximal input voltage allowed check your NI DAQ AI limits to set up this parameter This parameter is set up automatically but can be manually adjusted here if required Min minimal input voltage allowed check your NI DAQ Al limits to set up this parameter This parameter is set up automatically but can be manually adjusted here if required O ID the number of computer sound boards in the system This parameter is set up automatically but can be manually adjusted here if required Rate digitalization rate of the an
32. st THR the level to threshold norm histogram needed to determine the number of classes Digitalization rate is an important parameter indicating the number of samples per second of your acquired data Since this parameter influences many algorithms of data analysis and visualization we recommend paying particular attention to it UNIFE Section of Human Physiology 2012 FSPS User Manual Page 11 of 35 CO N 11 12 13 14 15 16 17 18 19 20 21 Peak width specifies the number of consecutive data points used in the least squares fit for finding peaks in the norm histogram Sensitivity an important parameter for adjusting the construction of the norm histogram thereby influencing the number of clusters determined Tolerated range is 1 8 3 3 Default is 2 6 the most common for various types of signals and acquisitions SPK show specifies the number of spike shapes to be visualized in each window of the Shapes Tab indicator Noise detect indicates if the detection of noisy spike shapes based on Mahalanobis distance evaluation 1s On or Off Control Tab 2 FCM status and performance information see Chapter 2 2 for details Information panel for number of clusters and features used Control button to RUN clustering classification algorithm Control button to RUN online classifier Control button to update several on screen parameters button performs screenshots capture of spike sor
33. ter Journal 1987 30 3 268 275 doi 10 1093 cominV30 3 268 for details SVD Singular Value Decomposition see here http en wikipedia org wiki Singular value decomposition for details UNIFE University of Ferrara http www unife it UNIFE Section of Human Physiology 2012
34. ting results currently visualized area of Control Tab 3 is captured and automatically saved to the same disk space where data files are stored Control button applies Mahalanobis Distance measurement to evaluate noisy spikes actually their PC scores located in the multidimensional feature space away from any class centre using probability density see p 14 Confidence interval for the Mahalanobis distance measurement Group indicator of data processing Control button to stop the program Control Tab 3 visualization and statistics See chapter 2 3 for details Information about detected and accepted spikes Squares at the top left corner of each graph are controls to change the colour of the class Sliding bar to navigate Raw signal graph 2 The indicator of the cluster s quality colour scale Chapter 2 1 Control Tab 0 Continuous or Active Triggered acquisition Continuous acquisition Triggered acquisition Raw signal Allows you to choose between continuous i Samples time this chapter and triggered Chapter 3 acquisition depending on your needs and experimental design UNIFE Section of Human Physiology 2012 FSPS User Manual Page 12 of 35 Chapter 2 2 Control Tab 1 basic I O parameters ess DAQ Tab options MAIN Filteri Spli S S E a i Ra O EO TO DO Detection SD value Threshold level is estimated DETECTION as SD value 6 1 10 Exponent Thr SDyat
35. tton IP in Figure 3 Scatter plots of Principal Component 1 vs Principal Component 2 for each spike waveform extracted automatically from the dataset 2D spatial distribution of PCs gives the experimenter an instant view of the maths of the recorded dataset and the consistency of trials within the experimental Condition UNIFE Section of Human Physiology 2012 es O O COND TAUON FSPS User Manual Page 29 of 35 Chapter 4 On line Spike Sorting Figure 4 15 14 13 P a o DEE Cj em 1 0 Parameters 5 I Channel oD ae k Dev1 ai do j Max Rate 3 o iif Lik Na 010 00 110000 all bl 2 i Mn samples ch o E 10 00 soo dk 0 2 A Controls 4 Classification TR A 5 i ON TT a TET a Sen TR Waveforms Gi 4097381 4122980 0 2 4 6 8 10 12 14 16 18 20 22 24 Time Time Sound Firing rate STAT Threshold sf 3713 1 C Programmi FSP5 _recordings 0 dat New q Original 80 4 Classified Co 3 Signal Inversion gt an HOD streaming Ci E E Class Class 3 l si 30 1 Classes Features pan 2 Legend 12 a Control group for Input Output parameters Classification control button to switch classification algorithm ON and OFF ON is default value 3 control button to switch visualization of waveforms in indicators 12 and 13 ON and OFF Switching OFF may
36. ue O THRESHOLD clusters standard deviation of the background noise SDyatue can N 4 Auto e gt a vary from 4 to 8 Default SDyatue is 7 A o pe features THRESHOLD New Keep is the control button New ei 3 Auto where O is an estimate of the noise allowing automatic New or manual Keep estimation of the threshold In the latter case you can adjust it by moving the cursor red line inside the Raw signal graph 3 New Keep allows the FCM clustering technique to make a new space segmentation and calculation of clusters centres New which is a necessary step of FCM unsupervised clustering technique Keep mode permits the classification of new objects and their membership values to the different classes for the given cluster centres which is used during classification procedure Auto Manual allows automatic determination of the number of clusters to be counted Auto Manual operation forces clustering algorithm to find exact indicated at the left side number of clusters The range is from 1 to 12 Auto Manual allows automatic determination of the number of PCs to be used during clustering classification Auto Manual operation allows user to manually choose dimensionality by inserting the value in the field next to the button Range is 1 to 5 determines the degree of fuzziness of the resulting clustering process As m 1 1 clustering tends to be fuzzy while m results
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