Worm analysis toolbox manual

18/12/2012 1 Worm Analysis Toolbox User Manual Version 1.0

1. Installation 2. Starting the analysis

a. Directory structure b. Vulva side selector

3. Preferences a. Head and tail calibration

4. Feature viewer 5. Worm diagnostics tool 6. Error window 7. Contact


The toolbox is compiled and tested for Windows operating system. To use the toolbox download and install the following: 1. MATLAB Compiler Runtime v. 7.14 Installing the runtime allows execution of the compiled Worm Analysis

Toolbox. It is for free and can be downloaded from Worm Tracker 2.0 website here.

2. ffdshow video decoder Ffdshow is a DirectShow and Video for Windows codec with support for a

wide range of audio and video formats, such as Xvid, DivX, and H.264. It will allow the toolbox to open worm video files. It can be downloaded here.

3. Microsoft Visual C++ 2008 SP1 Redistributable Package (x86) These libraries are required for successful communication between Matlab

Compiler Runtime and ffdshow.

http://www.mrc-lmb.cam.ac.uk/wormtracker/index.php?action=analysis

http://www.mrc-lmb.cam.ac.uk/wormtracker/index.php?action=analysis


1. Follow the installation wizard for the toolbox and click on the shortcut on the desktop of your computer. 2. In the main Worm Analysis Toolbox window click on Start to open analysis window. 3. In the analysis window click on Start to select a directory that will be analyzed. Behavioural features will be saved into a subdirectory “Results” and segmentation data will be stored in hidden directory “.data”. Overlay video will be saved in the same directory as the original video file.

1 2 3


1. Directory example with one experiment 2. Example of “.data” directory after the analysis. 3. Example of “result” directory – final output file containing results.

1 2

3


Vulva side assignment is essential for correct feature output. While head and tail is determined automatically vulva needs to be assigned manually. Add “_CW_” for clockwise or “_CCW_” for counter-clockwise from the head of the worm in the filename of the tracking experiment. For example:

N2 on food _CW_2012_05_09__11_52_00__1.avi gpc-1 on food _CCW_2010_03_09__15_13_17___2___10.avi


It is essential to make sure that the vulva side is recorded as seen in the video experiment.

It is most advisable to note down vulva side while entering a file name during the tracking process. Analysis toolbox will automatically assign correct vulva side and will ask the user to confirm. In case vulva side is not specified the toolbox will default to clockwise.


Redo stage motion diff: Recompute stage motion detection video frame difference data

Redo stage motion detection: Recompute stage motion using frame difference data

Redo segmentation Recompute video segmentation

Redo head and tail detection Recompute head and tail detection

Redo worm normalization Recompute normalized segmentation data. In case “Redo segmentation” is selected it will automatically trigger rerunning normalized data computation

Output video Output overlay video (save to file)

Remove overlay video Delete overlay video after completion

Calculate features Compute and output features for experiments

Standalone version Set toolbox process to be standalone. If disabled will use Schafer lab pipeline for analysis – not suitable for use outside Schafer lab

Database and NAS To be used in conjunction with the database pipeline in Schafer lab. Will have no effect in standalone version


Toolbox uses machine learning to predict head and tail of the animal. The algorithm analyzes motion and coloration data together with a training set assembled in Schafer lab to make a prediction. In case magnification and lighting settings are different in other laboratories the efficiency of the algorithm might decrease. Head and tail detection calibration allows to build a local training set to use for optimal head and tail detection. Use local head and tail calibration

Select this if you wish to use local training set Disable H&T warnings

Select the tick-box in case you don’t want to see warnings. Warnings will be produced in case the local training set contains too few data points of correct head and tail assignment

Calibrate Calls a function which takes directory containing head and tail assignment data as an input.


Calibrate 1. Create an empty directory that will store calibration files to create local head and tail assignment training set. 2. Copy a number of head and tail annotation files produced by the segmentation. There is one file created per experiment. (Example path for the annotation file: “C:\example\.data\N2 off food _CW_2010_04_29__16_31_42__5_seg\N2 off food _CW_2010_04_29__16_31_42__5_H&T.csv”). 3. Open both overlay video and head and tail annotation file for each experiment. In case there are incorrect h&t assignments in the video find video sequence in the annotation file by using the “startFrame” and “endFrame” columns. 4.In case the video segments indicated in the head and tail annotation file are assigned incorrectly – flip (if it was 1 write 0 and vice versa) the value in column “class” and save the file. 5. Do the same for all the segments and all the files.

chunkNo class cdfRatio1 cdfRatio2 cdfRatio3 cdfRatio4 cdfRatio5 tOrthoVStParaAbs hOrthoVShParaAbs validFrames posteriorReliability startFrame endFrame

1 0 1.12047 1.10059 1.10036 1.09253 1.06365 0.24124 0.44949 303 33 1 398

2 0 0.98547 0.99894 0.98691 0.97893 1.01647 0.60515 0.17522 746 25 399 1363

3 1 1.06655 1.01221 0.97691 0.9236 0.83187 0.31067 0.51684 145 5 1364 1560

4 1 0.99952 0.97631 0.96456 0.93241 0.85253 0.17261 0.60232 431 4 1561 2067

5 1 1.03775 1.0129 1.01599 1.01613 0.97003 0.21801 0.6579 313 18 2068 2475

6 1 1.04946 1.01863 1.01671 1.00613 0.94611 0.22464 0.46731 240 29 2476 2771

7 0 1.01533 1.03837 1.04192 1.06175 1.1249 0.50809 0.14343 459 6 2772 3314

8 1 0.96457 0.96103 0.9797 0.98853 0.96302 0.17752 0.46299 231 25 3315 3640

9 1 1.07276 1.05145 1.05015 1.04356 1.01408 0.24713 0.4768 1065 44 3641 5246


Data overview

• Expand feature groups to see time series data

• Explore annotation of the experiment


Time series view

• Explore the data by viewing the time series of a selected feature

• Select individual points to see the frame and data value


Worm shape view

• In case normalized data is available colour contour can be drawn. Change the toggle to revert to only skeleton view of the worm shape

• ROI toggle can show the worm as it is seen in the video or zoom in to bounding box around the shape


Histogram

• Histogram plots with associated aggregate statistics


Worm path view

• Worm path view indicating worms position on the path for the frame selected by the user in the time series view

• Darker path shows the path already travelled by the worm, lighter path is the path it will travel in the future


Playback controls

• User can playback the video and see the shape, the path and corresponding time series values for each part of the experiment

• Playback speed can be selected to either go through the frames quicker or slower


Worm diagnostics tool allows viewing the video to check if the segmentation algorithm is able to correctly segment the worm from the background. Detailed segmentation results are shown and different parts of the video can be checked.


It is important to ensure animals are in focus, not obscured by food or other objects on the tracking plate. Please ensure that the worm is dark on a lighter background and there are not artefacts like tracks or objects obscuring the worm. Worm diagnostics tool will indicate problematic frames.


In case an error occurs a pop-up window will display the issues encountered during the analysis process. Please use this to troubleshoot and include it when contacting us for support.


Tadas Jucikas [email protected] Eviatar Yemini [email protected] André Brown [email protected] William Schafer Group Cell Biology Division MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK. Tel: +44 (0) 1223 248011 Fax: +44 (0) 1223 213556

Worm Tracker 2.0 Website http://www.mrc-lmb.cam.ac.uk/wormtracker

Worm Behaviour Database http://wormbehavior.mrc-lmb.cam.ac.uk/

mailto:[email protected]









http://www.mrc-lmb.cam.ac.uk/wormtracker



http://wormbehavior.mrc-lmb.cam.ac.uk/



Documents

Worm analysis toolbox manual