Track-A-Worm - University of Connecticut Health Center
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1. 2 Click Connect Camera and wait a few seconds for the camera to start up and adjust The Camera indicator in the top right corner should turn from red to green 3 Enter the COM port which corresponds to the Prior Optiscan stage 4 Click Connect Stage and wait a few seconds for the stage to start up The Stage indicator in the top right corner should turn from red to green 5 Click Center to ensure that the stage is at its middle point 6 Enter the X and Y calibration values found in the Calibrate module 7 If necessary adjust the brightness auto exposure frame rate and recording time settings You must click Apply to send these settings to the camera The worm should be a dark grey color with good solidity easily distinguishable from the background If parts of the worm are too light and tend to blend in with the background Track A Worm will have a difficult time recognizing it 8 Note that there is a manual control for tracking threshold This is the brightness threshold which the software will use to differentiate the worm from background Typically Figure 5 Example of good lighting quality 80 is appropriate however if the image is very bright a higher threshold may be required For more information about selecting a threshold see the Playback section 9 Select the export folder for the recorded images the stage movement file will be automatically set 10 When you ar2. In addition to viewing controls Playback has a simple tool for cutting recordings The stage movement file must be entered into the file selection box because stage movement is also affected when frames are removed Then by entering a start and end frame and pressing Cut only the data selection is retained Note The lower cut edge should be a number of the form fn 1 where f is the frame rate and n is an integer The upper cut edge should be of the form fn This is because stage movement is tied to timing Therefore if the frame rate 15 and you want to keep data from frame 20 to 80 you should actually select from frame 31 to 75 The action of cutting creates a subfolder within the data folder called removed It also creates a copy of the original stage file appending _ orig to the original name The original unsuffixed stage file is cut to reflect the data selection Therefore all subsequent analysis should be carried out on the unsuffixed stage file The _ orig stage file and the contents of the removed folder can be used to manually restore the data folder back to its original state Export Movie The user can specify a range of frames to export and the file path to the movie File format is AVI and plays in standard media players such as Windows Media Player or VLC Be sure you have entered the correct frame rate before exporting If the box Show splines if available is checked and the splines are available
3. you will see a window pop up showing the worm with spline overlaid The movie will then be exported with the spline overlay Fit Spline The Fit Spline module takes the recorded images and the stage movement as input and produces spreadsheet files of spline point positions in x y pairs It requires some level of user input both to select the original threshold and to correct Track A Worm when it occasionally makes mistakes The inputs are both the data folder and the stage movement file In addition calibration frame rate and threshold must be entered The outputs are two spline files One contains stage compensated spline data meaning that the movement of the stage has already been accounted for The second suffixed with orig contains the raw spline data The raw unsuffixed data is typically more useful especially at high frame rates where the spline data may be incorrect due to inability of the stage to keep up with the frame rate Previously created splines can be reviewed using the appropriate input fields By default these file paths are set to reflect splines produced by the Batch Spline module However the user can elect to load any spline file by manually selecting them If ventral dorsal data was applied at the time of recording the ventral direction is indicated by a black arrow The head is represented by a red dot and the tail by a purple segment Fit Spline Worm Image F recordings 11202013ichr2 1 off Brow
4. 3 is selected the user clicks OK The window then transforms to show a bare blue spline The user then holds the Alt key and clicks the two points which roughly delimit the desired curve These markers can be dragged along the spline and can be deleted by right clicking The window will look like this 3 X 1028 s Y 915 3 7 C X 1097 Y 800 9 Curve 1 12 Curve 2 12 3 Curve 3 7 7 Curve 4 17 7 When the user clicks Done the final curve will look like this Curve 1 12 Curve 2 12 3 Curve 3 6 6 Curve 4 17 7 22 One last feature is that the user can simply select Do Not Segment which will fit a large circle to the entire worm body This is only useful when the worm is performing a thrashing behavior in which nearly the entire body forms a semicircle The typical workflow for using Curve Analysis is as follows 1 Select the spline file which the user wishes to analyze Either the unsuffixed or _ orig file can be chosen it does not matter Click Load 3 Either decide on a curve to analyze such as the head or decide to save all curves a If saving all curves simply click the respective radio button at the bottom of the window and proceed to saving the curve data b If selecting any particular curve continue below Enter the curve of interest into the box labeled Set all curves to and click Set 5 Using the Forward and Back button
5. If you enter the primary folder for any particular module Tif Export Folder for the Record Playback and Spline modules Spline Path for the Analysis modules and press Enter with the text field activated the other related fields will flash green for a moment and will update to reflect the standardized file locations Figure 2 Ventral directionality Calibrate Prior to making a recording the correct conversion factor from pixels to micrometers should be determined This is done by the Calibrate module calibration_gui Figure 3 Calibration interface The Calibrate module receives input from the camera Operation is as follows 1 2 Click Connect Camera and wait a few seconds for the camera to start up and adjust itself Place a calibration slide under the microscope and align it The slide should either be completely horizontal to calibrate the x axis or completely vertical to calibrate the y axis 3 Turn the magnification to the desired level 4 5 6 7 Click one end of the calibration slide and then the other end A line will be drawn across the image In the boxes labeled Calibration X and Calibration Y type the actual um distance spanned by the drawn line You may leave the unrelated window blank for example if you are calibrating in the x direction you can leave the Calibration Y box empty Click Done The results of calibration will be di
6. i7 PC but may take longer if the image is more challenging When the analysis is finished the splines will be displayed on the thumbnail images and the heads will be marked with X s Scroll through the thumbnails using the Up and Down buttons If you see a mistake it can be corrected as follows a Swapping If the head or tail was incorrectly recognized click Swap to fix it All subsequent thumbnails will also be swapped to compensate for the error checking algorithm previously described If you don t want this to happen simply swap the next frame so that the rest of the data returns to normal b Cross Omega shaped worms actually have four valid conformations which are in Swap any static frame indistinguishable to the Track A Worm algorithm O O The figure to the right 0 Q illustrates this concept 0 rH Notice how in this frame the head has a very small Swap Figure 8 Crossed and swapped conformations of an omega bending worm protruding segment which can complicate the differentiation of head from tail To overcome these issues Track A Worm references the previous frame to determine 1 whether the head and tail are in similar relative positions and 2 if the looped portion of the spline is progressing the same direction clockwise or counterclockwise Although these error checking mechanisms typically work correctly the interface provides the user with the ability to easily swap between all fou
7. is extremely important for Track A Worm The software expects to find files in a certain hierarchy if they are missing it can become confused A single recording is structured as such 1 Base Recording Folder i e wt 1 a Image Files img001 bmp etc i There may be a prefix R or L which denotes the ventral direction of the worm in relation to the head see figure below b Stage movement file wt 1 txt c Original stage movement file wt 1_orig txt this appears after you use the Cut function in the Playback module d Folder of removed images removed this appears after you use the Cut function 2 Spline file compensated for stage movement wt 1_spline txt this appears after you use the Fit Spline module 3 Spline file uncompensated for stage movement wt 1_spline_orig txt this appears after you use the Fit Spline module Note that there is only one base name wt 1 That is decided by the user during the recording process when choosing the Tif Export Folder The base name is echoed throughout the folder structure Also note that the spline file is not placed in the base folder but in the next folder up This is done to encourage the user to create a folder for recordings done on the same day which will contain all of the spline files and recording folders Track A Worm has functionality to preserve this folder structure while retaining some user control
8. Track A Worm Instructions and Notes Sijie Jason Wang MD University of Connecticut Health Center 2010 2014 Contents CO IC TIO ee te sancpasesesen cep sieayeteceuaniadutnsinsaiavasetacpsiegiatdeneatiatatuawsanenonodasstesen ide sastteteiepenadenosssnasconossesees A Few Notes about File SUUCTUN EG icccnccusdssrssvcsncnssvncranetvcaesnadnitedeasvine cnaavsoneessebccsuatniausnanwesn dnaveucesmumweaqsersunnane 91 ENE see en ee ee ee REC OT hse cera ere nace E te ete raat Nosesu E E ed eee BY OM Me ure ec nn cetaceans E E E E E A Fr Hs INA EE sion cene casas aietwosioss ceuc aoas cued aaa AA E E A AE EE AE EA PAU VAIN SUS EE E E E EAT A E A E E votsecsan T E AE A A A IEE A E 11 PEN FEIE GUEN onn E E A rer 12 ROOL MEINS GUATE era wmnnsindtaen eusunesseeudteen Gaubeaeesuseuienasunanmeteuitaencaueieesgeetades 13 NDO BEN Oe E E E E E S 13 SUMO AI BEN ea E E A E E A 14 TAL SAE a a T E E AR 14 Ted A D E a E EE E EE E E EE 14 IS CUO Mare E E 15 WOTA E e A T sent enn ens eanueec eaeoueasesse aes 15 NOSON 4 OE RES oc a E nee Or eee eee eon eee 16 BUC NAY SIS e E E A 17 Ex AU I NO A EEE EEES E E SENE PENEAN E EEEN 19 UNEARI SS EEE E E E aentoeaeoeee 20 Introduction Track A Worm is a MATLAB program which tracks the locomotion behavior of C elegans It was written in MATLAB R2013a using a Sony XCD V60 CCD FireWire camera and a Prior Scientific OptiScan Il moving stage It is composed of six distinct modules Calibrate Record Playba
9. ances of forward and backward motion Figure 14 Determination of Worm Direction Worm Amplitude Amplitude is another measure of worm curvature A highly curved worm tends to have a large amplitude and a straightened out one tends to have a smaller amplitude It is determined by first finding the velocity vector as described in the Direction section Then a box is drawn in alignment with the velocity vector and the width of the box is measured to be the amplitude The ratio of the amplitude to the length of the worm is also calculated Velocity Vector Ce Amplitude Figure 15 Determination of Worm Amplitude 15 Notes on Exporting Results There are two straightforward export functions Export Bend Data which exports the angle of every bend at every time point and Export Worm Path which exports the x y coordinates of chosen worm point cog or 1 13 for every time point For collection and analysis of large pools of data there is an Export Results subset of functions For any given recording the user may choosen an export file add comments if wanted and click Save and Append to export the summary of all analyses Because the Maximum Bend metric requires manual picking of peaks it may be disabled with a checkbox if the user wants to save time If the chosen export file does not exist it is created If it does exist the current data is appended to whatever data is already in the file Note tha
10. ck Batch Spline Fit Spline Analyze Batch Analyze and Curve Analyzer Together these components allow the user to determine worm behaviors including bending angle bending frequency movement speed and other characteristics Track A Worm is unpolished and contains some bugs There is still idiot proofing to be done As such it is important to follow the instructions in this document carefully Performing tasks out of order can disrupt the analysis procedure The program is launched by navigating to the program directory in MATLAB and typing launcher without the quotes Worm Track Launcher Determine the correct micrometer pimel Calibrate value for proper camera control and tracking R P Interface with camera and stage to record Pope worm movement Playback and manipulate recorded data Playback Find optimum threshold and video segment for spline fitting Select a group of video recordings to Batch Spline generate splines which may be reviewed corrected at a later time Track worm by fitting and reviewing Fit Spline splines Export spline data for analysis Analyze worm movement including angle Analyze direction frequency and other features Analyze worm movement in large batches Batch Analyze using a convenient GUI Analyze worm curvature radius by circle apie nazer fitting using the Kasa algorithm Figure 1 The Track A Worm launcher window A Few Notes about File Structure File structure
11. e satisfied with the settings manually move the stage to find the worm by hand not using the Align buttons make sure you keep the edges straightened out as you move it When the worm is within view press the Start button Do not manually move the stage after this point Note The Sony XCD V60 takes a few seconds to begin recording and you will have no control over it during this period Depending on lighting conditions you will likely see the autoexposure readjusting the camera for the first couple of frames As long as a small part of the worm remains in the field of view the worm can be reliably tracked However if the worm moves out of the field of view before the camera has settled you may need to cancel the recording described below Therefore the best way to begin recording is to move the stage so that the worm is just at the edge of the screen moving towards the center before pressing Start This minimizes the likelihood of losing the worm before tracking can begin 11 After the designated recording period is over Record will take a few seconds to save the files The message box at the top will indicate when the program is complete To begin a new recording you must press Connect Camera to restart the video feed and you must update the save paths Then you may start the new recording at Step 4 Canceling a recording If a recording fails you may cancel the session by clicking on the MATLAB command prompt
12. he centroid head and tail by typing O 1 13 into the box Calibration is entered simply by entering the x value followed by a space followed by the y value When all settings have been selected correctly simply choose the save path for the txt file and click Analyze and Export 18 Batch Spline The Batch Spline module allows the user to create a large list of recordings and run them as a batch This is particularly helpful when the user might be interested in leaving the software to run unattended such as overnight Simply click Add and select the folder threshold and framerate and click Analyze The files are saved in the folder with the names batchProduced_splineData txt and batchProduced_splineData_orig txt Once the analysis is complete the user can use the Fit Spline module to load these files and proofread them batchSpline_gui Batch Spline C Users Jason Dropbox challenging frames unc 9 2 T 100 FR 15 C WsersVason Dropbox challenging_frames unc 9 3 T 120 FR 3 C UsersVason Dropbox challenging_framesiunc 9 4 T 100 FR 15 C WsersVason Dropboxichallenging_frames unc 9 15 T 090 FR 15 C WsersVason Dropboxichallenging_frames wtS T 100 FR 15 3 6364 3 6364 Calibration Remove Folder Select Window Select a bmp folder Frame Rate 45 j 1 Threshold 100 19 Curve Analysis One unique feature of Track A Worm is the ability to determine the radius
13. hen play several seconds of footage If the worm remains a solid shape then the threshold is likely a good one Moving down in threshold tends to make a thinner more true to size worm profile This increases accuracy but also increases the chance of breaking the shape Moving up in threshold tends to make a thicker and easier to detect worm shape but can reduce accuracy In general you want to go as low in threshold as possible while retaining a valid profile This function takes some experimentation but is very easy to use after some experience Typically a good starting point is 100 140 Track A Worm uses a variable threshold algorithm which varies the threshold as much as 20 from the initial guess if a valid spline cannot be found Therefore a slightly bad guess does not necessarily mean the analysis will fail but will likely cause it to take longer Press Start Analysis After a moment the initial detection window will appear and you will be asked whether the head was detected properly The head is always marked with a cyan X The primary determining factor of head tail detection is the curvature of the ends However as a secondary error checking measure all subsequent head positions are referenced from preceeding values Therefore it is important to get a correct initial head position After dismissing the initial detection window the software will run on its own for a period of time Each frame usually takes 1 second on a Core
14. isplays the current frame and total number of frames The current frame can be selected by typing into the box Threshold Track A Worm uses thresholding which turns all pixels below the threshold value to black 0 and all pixels above the threshold value to white 1 Pixels can range from a value of 0 to 255 and typical effective threshold values range from 100 to 170 The output is known as a binary image Because thresholding is the first step in image analysis it is important to choose an appropriate value For very bright images a higher threshold should be chosen For darker images a lower threshold should be chosen Note that Track A Worm employs image processing techniques to remove background noise and fill in holes in the worm body Therefore even if certain parts of the worm are relatively light or if certain part of the background are dark Track A Worm will reject certain inconsistencies and still attempts to produce a viable worm profile Notice that in the above figure the thresholded worm image looks incorrect because the threshold has been set too low Lighter parts of the worm head and tail have been rejected as background because they were above the threshold limit Show Splines If Available Checking this box will overlay splines on the image if the splines have already been produced and reside in the standard location see A Few Notes on File Structure Apply Apply changes to threshold and frame number
15. l Bends is a useful metric for quantifying the bending behavior of an entire worm It is simply the average of sum of all 11 bends of a worm across all measured times Note that unlike the the standard bend trace the Sum of All Bends metric uses the absolute value of the bends so the direction of the bend is not considered Thrashing Some worms move in a vigorous whole body flipping motion called thrashing This analysis counts the number of thrashes and produces the frequency of thrashing as well The metric is produced by fitting a circle to the entire worm shape and noting the number of times this circle flips from ventral to dorsal or vice versa Speed and Distance The Speed function measures the average speed a worm moves in m sec the Distance module measures the total distance a worm moves in um 14 Direction The Direction function measures the number of frames in which a worm is moving forward and the number of frames it is moving backward Directionality is determined by the velocity vector the vector from the centroid of the current frame to the centroid of the next frame Then the head vector is defined as the vector from the current centroid to the current head point If the vector projection of the head vector onto the velocity vector is positive the worm is determined to be moving forward If the projection is negative the worm is moving backward In addition the Direction function outputs the total dist
16. lar to below wa rame cuv V D 1 Curv V D 2 Cuw 3 V D 3 Curv 4 V D 4 Cumw 5 V D 5 Cuw 6 V D 6 1 0 13771 D 0 17273 V 0 18973 D z E 2 0 524 V 0 12623 D 0 16984 V 0 2735 D 3 0 27793 V 0 13994 D 0 17251 V 0 24049 D 4 0 18885 D 0 17885 V 0 25384 D 5 0 5541 V 0 15182 D 0 16739 V 0 21954 D 6 0 25552 V 0 17259 D 0 156 V 0 21737 D 5 5 3 7 0 18954 V 0 18255 D 0 17169 V 0 22829 D 8 0 12778 V 0 19016 D 0 17879 V 0 21495 D a n1192 wW n1776 M NIARIT n anna N N e 5 Notice how each curve has a subsequent column showing whether it is ventral or dorsal in direction Occasionally this module can produce erroneous curves which do not visually match This is usually because the worm is making an unusually tortuous bend and therefore while the spline might be divided cleanly by inflection point the segments delineated by the inflection points might not actually be a circular shape There is a special feature in the Curve Analysis module which allows the user to re assign the limits of the curve For example the user might see the following spline Curve 1 12 Curve 2 12 3 Curve 3 7 7 Curve 4 17 7 21 Here the problem is that curve 3 really does not correctly approximate the smallest part of the curve due to the elongated shape of that particular curve The user can then click Fix curve which produces the following window Enter curve to modify Once curve
17. le and calibration information are required for this subsection as well as the spline point to analyze 1 13 If ventral dorsal information has been recorded all angles will be reported with the convention of positive being ventral and negative being dorsal Otherwise polarity is arbitrary 11 Analysis General Settings Spline Path F recordings 11202013 chr2 1 off_spline_orig txt Browse Frame Rate 15 Note Choose the _orig export file not the unsuffixed standard file all Frames to Analyze Bend Analysis _ Movement Analysis Stage Path F wecordings 11202013 chr2 1 offichr2 1 offtt Browse Bend Number 1 11 1 ss a Spline Point 1 13 or 0 for centroid 0 x 3634 Y 3 6364 Plot Bend Trace Sum of All Bends i Dominant Frequency and RMS Maximum Bend o Plot Worm Path a Worm Amplitude Thrashing Speed and Distance Direction Metrics Export Bend Data a a 2 ae ae Export Worm Path Messages A _ _ _______ Messages Export Results Export File F temp tet Comments Manually Produce Maximum Bend During Export Save and Append Figure 10 Analysis window One useful feature is that the user can specify only certain frame ranges to analyze determined from the Playback module For example it is often useful to only analyze linear movement The user would need to find the range of frames that the worm is moving linearly and then enter those values int
18. o the Analysis module The correct format is f1 f2 i e 31 80 without the quotes Otherwise enter all to analyze the full worm path In addition to full exporting of all bending and movement data the following analyses are currently built into Track A Worm Bend Frequency The Frequency analysis measures the frequency of the primary component in any given bending trace The bending trace is simply the plot of bending angles as a function of time For example in the below figure a primary frequency of approximately 0 5 Hz dominates the bending behavior Smaller higher frequency components are also present but are ignored by this metric Occasionally 0 Hz may be identified by the software as the dominant frequency This is erroneous and therefore the algorithm rejects all results below 0 1 Hz and will pick the next highest peak Use of this metric will also produce a plot of the frequency spectrum for verification 12 Figure 11 Bending Angle Bend Trace Frequency Spectrum of Head Bending Trace oo on So 60 tuo o So 40 i mm ha n So hJ o oS i mn 2 m Bending Angle Adeg a Magnitude ka co am Z mm iis 500 60 O 0 2 4 6 3 10 12 14 1 2 3 4 5 6 7 Time sec Frequency Hz Figure 12 Example of a head bend trace and the corresponding frequency spectrum Root Mean Square Root mean square RMS is a measure of the overall magnitude of a signal A
19. of individual curves formed by the worm It first reconstructs a smooth curve from the 13 point spline Next it determines the inflection points of this curve Next it excludes segments which are shorter than a predetermined length in order to remove artifactual curves Finally it determines a best fit circle for each bend and returns the radius of this curve as a percentage of the total length of the worm Curves are numbered from head to tail The key concept of this module is that the curve of interest for the user may sometimes change in its numbering This is because the software simply numbers curves from head to tail For example the user might be interested in the head bend which will typically be the first bend However in some frames that bend might be displaced and change in numbering Therefore the Curve Analysis module is designed so that the user can first select the general curve number that he is interested in and then scroll through the frames to make corrections as needed In the below example in the bottom right frame notice that the head curve is now actually curve 2 The curve selection box has been corrected accordingly The user needs to press Enter after changing the number in order to register the change Sometimes the extra curve at the head is not actually erroneous but is actually a new head curve which is propagating forward This is left up to the user to determine how to best assign curves Curve Analy
20. pplied to the head bending trace a higher RMS indicates a greater magnitude of bending The formula used is where n is the total number of points and i refers to each individual point RMS is often a very sensitive way to differentiate phenotypes but its drawback is that a worm with very chaotic small bending movements could produce the same RMS as one with more organized large bends Maximum Bend Although worms display complex behavior particularly in the head it is often useful and intuitive to find the maximum sweep of a bend The Maximum Bend analysis allows the user to place markers at the maximum and minimum points of excursion in each cycle of a worm bending trace The analysis module 13 subtracts the average of the most negative bends from the average of the most positive bends The user must hold down the Alt key while clicking peaks and troughs alternatively he or she can right click and select Create New Datatip for each point Worms with very chaotic bending behavior can cause this tool to become unreliable since the user must be able to visually distinguish extrema MEE Hold the Alt key when clicking the graph to create cursors E i oe ee X 1133 Y 5616 1087 Y 54 38 Ix 6 933 Y 53 44 4 S67 Y S141 37 Be 51 06 B X 0 3333 X 1 867 TE E Y 56 46 Finish Analysis Figure 13 Bend trace showing markers for the points of maximum bend Sum of All Bends The Sum of Al
21. r conformations if there has been a mistake c Manual Clicking the Manual button will open a new window displaying an enlarged version of the thumbnail of interest You have two options here You can either draw an approximate spline or you can try to have Track A Worm find a spline with a new Threshold value To draw a manual spline click Start and then click at least 9 consecutive points along the midline of the worm Your points do not need to be equally spaced but they do need to be accurate When satisfied click Done and the spline points will be placed To try a new automatic spline simply enter a new threshold value and click Adjust As before there is some leeway here because Track A Worm will shift the threshold as much as 20 to find an Adjust Threshold appropriate spline 100 dnt 8 Once you have scrolled through and checked Figure 9 Manual spline creation all of the thumbnails click Save Changes to write all data to the spline file Analysis The Analysis module includes several data analysis tools as well as raw data export tools It is separated into three subsections General Settings Bend Analysis and Movement Analysis The text entry fields in the General Settings must be filled prior to any analysis Bend Analysis functions require you to input the bend you want to analyze 1 11 Because distances are applicable to Movement Analysis the stage movement fi
22. s scroll through the recording and correct any erroneously labeled curves by either changing the number be sure to hit Enter after changing anything or by using the Fix curve button 6 When satisfied with the curve selections click Save Curve Data and choose the output file path 23
23. s up a small window to select spline file stage file and frame selections 17 fileSelect Window x Spline and Stage selection k Documents and Settings Zhaoven WangiDesk Browse c Documents and Settings ZAhaowen Vang Desk Browse Frame Selection 1 50 Done As in the other modules stage selection is performed automatically when the spline file is chosen If a change is made to the text box pressing Enter will flash the stage box green indicating that the selection has been updated The Browse button accomplishes the same thing Frame selection is performed using the same syntax as the standard Analysis module enter 1 50 to select all data between frames 1 and 50 or enter all to select the entire recording Do not include the quotes When removing and editing recordings a dialog box will prompt the user for the recording number corresponding to the list entry Analyses are chosen via checkbox A few options are also required Bend Numbers are chosen using standard Matlab syntax You can choose individual bends using the format 1 3 5 indicating discrete points You can choose entire ranges of bends using the format 1 5 indicating a series of connected points Remember that there are a total of 11 bends for a worm spline Point numbers are also chosen using this syntax However the choice 0 is used in this context to select the centroid of points For example you could select t
24. se Stage Data F recordings 11202013 chr2 1 offichr Browse Load i ANALYZING IMAGES FOR REVIEWING AND CORRECTING PREVIOUSLY CREATED SPLINES ie batch produced F lrecordings 11202012 chr2 1 off_spline_orig bet eee Calibration 3 6384 3 6384 Frame 15 Thresho 100 Start Analysis ane Dain See ee ee eee if ventral direction wes recorded it will be denoted by arrow Spline Data FaArecordings 1120204 3 chr2 1 off_spline txt Browse hag ol Manual kI kir k Cross E Cross kI Cross kir gpi Cross Omega aa a Swap Manual Manual Manual swap Manual Swap Manual Swap Manual Manual Cross Omega Cross Omega Cross Omega Cross Cross Omega Cross Omega Cross Omega Cross Omega Cross Omega Spline FArecordings 1 120201 3 chr2 1 off_spline txt Browse Save Changes Figure 7 Spline fitting interface Operation is as follows 1 Select the desired stage movement file data folder and spline export file 2 3 4 5 6 7 Press Load a If reviewing previously produced splines enter the file paths to the orig and unsuffixed spline files in the appropriate boxes and press the corresponding Load button Enter appropriate calibration and frame rate values Use the Playback module to find an appropriate threshold value It is usually a good idea to select an approximate threshold and t
25. splayed in the Results box Only keep the calibration for the axis you are concerned with Click Disconnect Camera before exiting the module Record The Record module receives input from the camera It outputs stage movement information to the OptiScan stage and to a user designated txt file It outputs sequential images comprising the recorded movie to a user designated folder Connect Camera Disconnect Camera i Record Time sec 30 Apply Connect Stage COM 8 Disconnect Stage Select Bmp ex folder CAUsersiS Jason Wang Desktopivwt 1 m Align Stage _______ pon op Browse ere Select export tor stage movement CAUseraiS Jason WangDeskopwi witii ww e center gt gt V Lv Calibration um px Tracking Threshold x 3 6364 3 6364 Start Ventral right Ventral left Abort Ventral unsure Status Panel Stage Waiting Optogenetic Stimulation Connect NIMyDAQ Disconnect DAQ ent o 0rF 1 0n x 1 ch2 0 0FF 1 0N s e Echa o orF 1 0n x 4 Show Sequence Graph Sequence length 9 sec Figure 4 Recording window A typical workflow is 1 Ensure that the camera is connected via FireWire 800 and that the stage controller is connected via the serial port on the right side when looking at the computer from behind Ensure that the stage controller is turned on
26. t it is very important not to modify the export files manually using Excel Notepad or any other application until you are completely done This prevents corruption of the formatting 16 Batch Analysis The Batch Analysis module contains all of the previously described analysis tools but offers a user interface that is better suited to analyzing large sets of data Recordings are added via a small spline file stage file frame selection interface to an overall list of files batchAnalyze_gui ssi Batch Analysis 13 0owen angDesktopyyormtrackerrecordings 7 7 10nx16 1 spline orig txt WZhaoven Yvang Desktop ywvormtrackeryecordings 7 104nx18 1 nx1 8 1 txt all 2 oVvwen YvangiDesktopwrormtrackerrecordings 7 7 10inx18 2_spline_orig txt WZhaovwen Yvang Desktop wyormtrackerrecordings 7 7 10 nx18 2nx1 8 2 txt 1 30 Add Recording Remove Recording Edit Recording Clear List Analyses lw Avg Sum of Bends mM Avg Amplitude M Frequency MV Avg Speed Iv RMS I Total Distance lw Avg Bending Range I Direction Metrics Options 3 Framerate 0 5 Point Number s 1 11 Bend Numbers 5 5364 3 Calibration Old Tracking Style Pre 6 4 10 Save Path keDocuments and Settings Zhaoven Vyang iDesktapy Browse Analyze and Export yy ll The list is manipulated with the Add Recording Remove Recording Edit Recording and Clear List buttons Recording and editing recordings bring
27. to make it the active window and then pressing CTRL C A series of error messages will appear and the recording will be canceled You will then need to click Disconnect Camera and then Connect Camera This returns you to Step 4 Playback The Playback module is extremely useful for looking back through data selecting relevant portions to analyze and searching for proper imaging thresholds explained below Playback Playback Control Frame Pause 15 Play Frame CWsersis Jason Wang Dropbox challenging_frames unc 9 Browse Load 1 45 Cappy Threshold 55 Use Thresholded Image Show splines if available Frame Selection C Users s Jason Wang Dropbox challenging_frames unc S Browse 4 4 Cut Reduce Frame Rate Export Movie Frames 1 Enter howie Path Browse Export Mowie Figure 6 Playback module showing a thresholded image In order to load a recording browse for a data folder using the file selection field under Playback Control Then press Load to open the recording Playback Control button functions are as follows Frame Frame Move forward or backward a single frame Pause Play Frame Rate Control playback The Frame Rate control between Pause and Play determines how quickly the recording will play The actual playback framerate depends on the speed of the computer so the values are not true rates but can be adjusted in relation to each other Frame Control This d
28. zer Select spline file C UsersVason Dropbox challenging_frames v l Browse Load Do Not Segment Set all curves to 1 Set Solid circles ventral Dashed circles dorsal Dotted circles either Frame 21 405 Frame 22 405 Curve 1 15 3 Curve 2 19 6 Curve Curve 1 12 7 Curve 2 19 9 Curve 3 18 1 Curve 4 21 3 1 3 19 6 Curve 4 55 1 Fix curve Fox curve Frame 23 405 Frame 24 405 Curve 1 12 6 Curve 2 20 Curve 3 Curve 1 41 9 Curve 2 15 2 Curve 16 7 3 19 9 Curve 4 24 7 Fx curve Fox curve Back 21 24 i Forward gt Save selected curves Save Curve Data Save all curves 20 Here is what the above selection would produce as an output if the user chooses Save selected curves Frame Curve Radius Length Ratio Ventral Dorsal 21 1 0 15309 D 22 1 0 12677 D 23 1 0 12631 D 24 2 0 15219 D Notice that at the bottom of the window the user can actually choose between two radio buttons If Save selected curves is selected then the user must be sure that he has actually assigned curves by either manually going through the recording or entering a curve number in the box labeled Set all curves to and clicking Set If there are frames remaining which do not have curves assigned the program will throw an error The other radio button is Save all curves which will simply output every single curve in a large spreadsheet appearing simi
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