Tracking intracellular vesicles in cells

Hello community,
I need help regarding tracking of intracellular vesicles in cells. I have played around a bit with Particle Tracker 2D/3D included in Mosaic Toolsuite. I think i manage to track particles to some extent. But, i am struggling to get track length and velocity for each particle. I will appreciate any help/advice/suggestion.
I could not upload the multiTIFF image. Here is dropbox link:
I have attached herein the first frame of the file. What I am aiming to do is to track the vesicles (total 60 frames) and obtain track length and velocity of individual particles.
cell1-2.tif (528.1 KB)
Thank you so much for your help.

Hi @sphuyal and welcome to the forum!

I believe you could get good results using the well documented TrackMate in Fiji. I used it on your example cell1 and got some encouraging results concerning spot detection

I think if you follow the documentation you should be able to calculate the features you are looking for

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Thank you so much @CellKai. If it is not too much work, could you please share some of your parameters that you used in TrackMate? It will help me immensely. I am a total novice in tracking and was overwhelmed by the options in Trackmate.

sure, no problem.
The first thing I noticed is that your image scale for “cell1.tiff” seems incorrect and is 0.0000023 micron/px with a frame interval of 0 seconds, so I think you should manually update the spacial calibration of the image. Since I did not know the real values, I set it to 1 micron/px and 1 second/frame by selecting the image and editing its properties in the Image > properties dialog.

Then I started Trackmate and continued next with the below settings:

  • select a detector: Log detector

  • select a view: Hyperstack Displayer
  • Set filtes on spots: quality above 9.38 AND mean intensity below 420.34
  • Select a tracker: simple LAP tracker
    • here it would be important for you to consult the documentation on what “linking max distance”, “gap-closing max distance” and “Gap-closing max frame gap” refer to so you can choose meaningful values for your experiment. I just used the default values.

Now the tracks are created and you can set for example a couple of filters concerning their velocity or duration.
in the next menu you have the option to view all track statistics by pressing “Analysis”


the final menu lets you export the spot statistics or all tracks and also lets you capture an overlay, i.e. a movie of your spots following the traces.

Hope this helps!


Thank you very much @CellKai! I am truly grateful to you for this support!