Organoid Tracker Usage

Step-by-Step Protocol for Using Organoid Tracker

Manual tracking: 

1. Open your images: 

File > Load Images/ Load tracking data 

Supported formats: TIFF, TIFF series, LIF, CZI, ND2 

2. Editing positions:

• Edit > Manually change data or press C

• To insert a position: press Insert/Enter.

• To delete a position: press Delete/Backspace.

• To move a single cell to the mouse position: select it and hold Shift.

• Navigate timepoints: use left/right arrows.

• Navigate z-direction: scroll the mouse wheel.

3. Linking cells:

• Select a single cell.

• Move to the next or previous timepoint.

• Press Insert to create a link to the new position

4. Working with many positions:

• Select > Select positions in a rectangle or press R. 

• Move positions:

      -Alt + A/D: left/right.

      -Alt + W/S: up/down in Y.

      -Alt + Q/E: up/down in Z. 

• Delete selected positions: Ctrl + Delete 

Automatic tracking:

Before you start, create a directory in which to store all the results of your analysis. 

1. Obtain nucleus positions: 

• Open OrganoidTracker and load your images. 

• Go to: Tools > Detect cells in images.

• Select the appropriate neural network (pre-trained or trained locally).

• Create a folder for detected positions.

• Run the script:

organoid_tracker_predict_positions.bat

• You have a folder called “Automatic positions” that contains a .aut file. Load it via File > Load tracking data. 

2. Obtain division likelihoods: 

• Go to: Tools > Detect division likelihoods. 

• Select the trained network for cell divisions

• Create a folder called Division.

• Run: 

             organoid_tracker_predict_divisions.bat

• The folder Division predictions will contain the results.

3. Obtain link likelihoods: 

• Go to: Tools > Detect link likelihoods. 

• Select the trained network for links.

• Create a folder Links.

• Run: 

            organoid_tracker_predict_links.bat

• You have a folder called “Link predictions”. 

4. Create tracks: 

• Go to: Tools > Create tracks. 

• Create a folder Tracks. 

• Run:

             organoid_tracker_predict_tracks.bat

• The folder Output tracks will contain your tracks. 

5. Calculate error rates: 

• Load images and Final link - clean.aut. 

• Go to: Tools > Compute marginalized error rates.

•  Select “All possible links - clean.aut”.

•  Create a folder “error_rates”.

• Run: 

             organoid_tracker_marginalization.bat

• The output will be Filtered_positions.aut or Solution_link_file.aut.

6. Manually correct warmings: 

• Load images and Solution_link_file.aut.

• Go to: Edit > Manually change data.

• Correct errors using the same controls as in manual tracking.