Worm Analysis Toolbox 1.0: User Manual



Worm Analysis Toolbox 1.0: User Manual



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7/16/2009

Tadas Jucikas, Victoria Butler and Eviatar Yemini

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH

UK.Tel: +44 (0) 1223 248011 Fax: +44 (0) 1223 213556

Web:

Contents

Contents 2

Table of Figures 3

Introduction 4

Installation 5

Starting the Toolbox 6

Key Features 7

Segmenting the worm 8

Feature Extraction 13

Creating a directory 13

Skeleton alignment and orientation 15

Calculating metrics 21

Drawing histograms 24

Helper Tools 26

Worm Viewer 26

Video Diagnostic Tool 28

References 30

Table of Figures

Figure 1 Main User Interface 6

Figure 2 Module 2 Segmentation Interface 8

Figure 3 File browser for loading videos for segmentation 9

Figure 4 Module 2 Segmentation Interface displaying segmentation result 10

Figure 5 Module 2 Segmentation Interface indicating segmentation of the video is complete 11

Figure 6 Module 3 Feature Extraction and Analysis Interface 13

Figure 7 File browser for selecting folder for directory structure creation 14

Figure 8 Loading worm skeletons for alignment and orientation checks using Module 3 Feature Extraction and Analysis Interface 15

Figure 9 File browser for selecting folder containing Worm* folders to check skeleton orientation 16

Figure 10 Wormproc window for checking skeleton alignment and orientation 17

Figure 11 Worm duplicate check box 17

Figure 12 Wormproc window displaying worm skeleton 18

Figure 13 Dialogue box to save aligned worm skeletons 19

Figure 14 Status box in Module 3 indicates skeleton alignment completed for a folder 20

Figure 15 Entering calibration factors to calculate millimeters per pixel 22

Figure 16 Status of Module 3 Feature Extraction and Analysis indicating metrics are being calculated 22

Figure 17 Status of Module 3 Feature Extraction and Analysis indicating that metrics have been successfully calculated 23

Figure 18 File browser for loading folders to plot histograms 24

Figure 19 Module 3 Status box indicating histogram function failure 25

Figure 20 Worm Viewer Interface 26

Figure 21 Worm Viewer Interface with video and skeleton successfully loaded 27

Figure 22 Worm Diagnostics window 28

Figure 23 Worm Diagnostics window with video successfully loaded 29

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Worm Analysis Toolbox 1.0: User Manual

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Introduction

Worm Analysis Toolbox 1.0 is a free worm video data analysis package. It was designed to be used with data collected with Worm Tracker 2.0. It is a first beta release programmed in Matlab. Source code will be available soon. At the moment please download compiled Worm Analysis Toolbox version.

The software was created by Tadas Jucikas and Eviatar Yemini and is tested by Victoria Butler at the Schafer Lab. Please send an email to Tadas in case of technical issues, suggestions and bug reports. Send an email to Victoria with usage questions and experimental issues.

Installation

You will need Matlab or Matlab Compiler Runtime Environment (MCR) to run Worm Analysis Toolbox 1.0. Installer will automatically download and install MCR if you don't have Matlab. If you want to install the toolbox on a computer disconnected from the Internet, please download the MCR from here and install it manually. You will need to download and install ffdshow a media decoder used for decoding compressed video formats. You can download it from our website, or look for the newest version in ffdshow website.

Starting the Toolbox

• Open the Worm Analysis Toolbox, either from the Program menu or by double-clicking on the Worm Analysis Toolbox shortcut.

• This will open the main user interface shown below:

[pic]Figure 1 Main User Interface

Key Features

• The main user interface has 6 buttons; 3 modules and 3 helper tools

• The 3 modules are:

1. Module 1: stage movement

This button is currently not functional

It will allow the path of the worm to be recreated and viewed

2. Module 2: segmentation

This button allows the user to load collected videos for segmentation of the worm

3. Module 3: feature extraction

This button allows the user to load segmented worm skeletons for feature extraction

• The 3 helper tools are:

1. Worm Viewer

This button allows the user to load a video and the segmented worm skeleton and view the overlap between worm and skeleton

2. Worm Diagnostics Tool

This button allows the user to load a video and check the segmentation result

3. Dropped Frame Detector

This button allows the user to identify frames dropped by the segmentation process

This button does not need to be used in the latest version of Worm Toolbox

Segmenting the worm

Once videos have been collected with Worm Tracker 2.0 software they can be loaded for segmentation. This process identifies the worm in the video and distributes a skeleton of 13 points along the length of the body.

To segment collected videos:

• Click on the “Module 2: segmentation” button on the main user interface.

• This will open the “Module 2: Segmentation” interface:

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Figure 2 Module 2 Segmentation Interface

• Click the “Load” button at the top right of the interface.

• This will open the following file browser:

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Figure 3 File browser for loading videos for segmentation

• Select the folder containing the videos to be segmented and then click “OK”. The folder name will be loaded into the bar in the “Control panel” box.

• Click the “Start module 2” button.

• After a few seconds, the first video will be loaded and segmentation will start. The video frame with the segmentation result can be visualized in the “Segmentation result example” box. The segmentation result is shown for every 200 video frames.

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Figure 4 Module 2 Segmentation Interface displaying segmentation result

• The status box indicates:

1. The video currently being analyzed out of the total number of videos loaded

2. The frame currently being analyzed

3. The status of the segmentation – whether segmentation is still ongoing or segmentation of the videos is complete

• The “Width profile” box displays the width of the worm from the calculated skeleton; from the head to the tail down one side of the worm and then from the tail to the head down the other side of the worm. This graph should ideally be symmetrical about the middle of the x-axis, indicating the skeleton is positioned centrally on the body of the worm.

• Once segmentation is complete the “Status” box will indicate that “Module2 finished analyzing files in …” the loaded folder and the module 2 interface can be closed.

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Figure 5 Module 2 Segmentation Interface indicating segmentation of the video is complete

• The folder containing the loaded videos will now contain the successfully segmented worm skeletons as _seg files, preceeded by the date and time each video was collected. Copy these _seg files into a new folder.

• The folder loaded will also contain 2 new folders called “finished” and “unfinished”. The “finished” folder will contain all videos successfully segmented. The “unfinished” folder will contain videos that were not successfully segmented.

• If segmentation of the video is unsuccessful the “Status” box will indicate that “Module2 finished analyzing files in …” the loaded folder a few seconds after clicking the “Start module 2” button.

• Unsuccessful segmentation most likely results from the first video frame containing the worm coiled on itself in some way. Ensure that videos are started when the worm is moving forward and the entire length of the body is clearly visible.

Feature Extraction

Creating a directory

Before continuing with analysis of the segmented worm skeletons, a directory structure must be constructed for the worm videos to sort videos of the same name into the same folder.

To do this:

• Click on the “Module 3: feature extraction” button on the main user interface. This will open the “Module 3: feature extraction and analysis” interface:

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Figure 6 Module 3 Feature Extraction and Analysis Interface

• Click on the “Start Create” button at the top right of the interface in the “Step 1: Create dir structure” box

• This will open the following file browser:

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Figure 7 File browser for selecting folder for directory structure creation

• Click on the folder that contains the _seg skeleton files and then click “OK”.

• After a few seconds the “Status” box will indicate that a new directory structure has successfully been created.

• This new directory will sort all worm skeletons with the same assigned name into the same folder and number them worm 1 to worm X where X is total number of videos with that particular assigned name. It is therefore important to keep the naming of videos consistent.

• The organized folders can then be loaded to check skeleton orientation.

Skeleton alignment and orientation

The “Module 3: feature extraction” button also allows the user to check the segmented worm skeleton, define the head-tail orientation of the worm and view and edit excluded frames.

To do this:

• Click on the “Load” button in the middle of the “Step 2. Wormproc: alignment and orientation” box:

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Figure 8 Loading worm skeletons for alignment and orientation checks using Module 3 Feature Extraction and Analysis Interface

• This will open the following file browser:

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Figure 9 File browser for selecting folder containing Worm* folders to check skeleton orientation

• Click on the genotype folder containing the segmented worm skeletons to be analyzed and then “OK”.

• This folder name will then be loaded into the bar at the left of the “Step 2. Wormproc: alignment and orientation” box.

• Click the “Start Wormproc” button at the right of the “Step 2. Wormproc: alignment and orientation” box.

• If the folder is being loaded into Wormproc for the first time, this will open the Wormproc window, shown below:

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Figure 10 Wormproc window for checking skeleton alignment and orientation

• If a folder has been loaded into Wormproc before, the following “Wormproc duplicate check” box appears:

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Figure 11 Worm duplicate check box

• If you want to overwrite the previously saved head-tail orientation for a particular worm video click “Yes”, if not click “No”. When “Yes” is clicked the Wormproc window is opened. If “No” is clicked a “Wormproc duplicate check” box appears for the next worm video in the folder.

• To visualize the worm skeleton click the “Play >” button at the left of the Wormproc window. The skeleton of the worm will appear with 13 points connected by blue lines. A red circle at one end of the skeleton identifies the head.

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Figure 12 Wormproc window displaying worm skeleton

• The playback speed of the video can be adjusted using the “Playback Speed” bar at the bottom of the Wormproc window.

• The playback speed can be further increased or decreased using the fast-forward (“>>” and “>>>” buttons) or rewind buttons (“ ................
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