Exercise 4 - 3D segmentation using TrackMate (StarDist)

3D stack of cells in a spheroid from Zenodo.

Download TIF file

Open above Z-stack in Fiji and run the TrackMate plugin, just like in Exercise 2.
Since this is a Z-stack and TrackMate works on a time-lapse sequence, we need to swap Z and T dimensions. TrackMate automatically detects it and asks for dimension swapping. Click Yes.
For the detector, choose StarDist detector from the drop-down menu.
For the tracker, choose Simple LAP tracker and the following tracking settings:
Linking max distance=5 pixel (a lower value than in Exercise 2, since the cells are not moving)
Gap-closing max distance=10 pixel
Gap-closing max frame gap=0 (since cells are not disappearing from frame-to-frame)
Keep clicking the next button until you reach the Display options window (image below). Choose:
Color spots by: Track index
Uncheck Display tracks

Every cell will be outlined in a different color. Scroll through the stack to check the accuracy of the results. If results are not optimum, go back to the detection and/or tracker steps by clicking on the previous button (green left arrow) and changing the settings under detector and tracker.
On the last TrackMate window called Select an action, generate a label image by selecting Export label image from the drop-down list and clicking Execute. It will generate a grayscale Z-stack.
Apply colors to cells with an LUT: Image › Lookup Tables › glasbey_inverted
For creating a 3D rendering, swap Z and T dimensions back to the original values by selecting Image › Properties... and entering Z=64 and T=1. Click OK.
Generate 3D rendering by using 3D viewer plugin: Plugins › 3D Viewer. Select Resampling factor = 1.
If a window pops up asking to convert the Z-stack to 8-bit or RGB image, click Yes.
In the ImageJ 3D Viewer window, use left mouse click and drag to rotate and inspect the volume.

Well done, if you finished all 4 exercises!