Problems using the CASA plugin to successfully track cell movement

I have been having some issues with the CASA plugin for ImageJ (

I am unable to get it to correctly track the sperm cells in my TIFF stacks I have taken of sperm cell movement.

Currently I am applying a threshold to my TIFF stacks so the cells appear black on a white background.

When using the CASA plugin I am leaving the most of the settings for sperm motility as the default settings.
But adjust the pixel size range for the cells, minimum frame length for tracks, maximum sperm velocity between frames (pixels) and the framerate at which it was recorded.

I’ve measured some of the sperm cells so they appear to be between 10 to 15 pixels in diameter so I have those settings at minimum of 7 pixels and maximum of 20 pixels.
As most my TIFF stacks are roughly 30 frames or higher I have the minimum track length set to 30.
I have done some rough calculations that the cells are moving roughly 50 to 60 pixels per frame from there previous position. So have set the max velocity to 100 pixels. Increasing further will eventually lead to the plugin thinking 2 separate cell on opposite sides of the field of view are the same. Resulting in very messy and incorrect tracks. The framerate recorded at should be 120 fps so I have set the frame rate to 120.

Interestingly I had this work with these settings and got some really nice tracks from one of the videos (converted to TIFF stacks for analysis with ImageJ) I took a couple of days ago. However I since then I haven’t experienced the same success on other videos and now can’t replicate the same tracks on the original TIFF stack with the same settings.
Additionally I am now also having issues where if I change to many of the measurement variables on the CASA plugin it won’t actually finish processing the TIFF stack and just stops. Resulting in on track image or motility data.

Does anyone with some experience using this plugin or tracking cell movements have any suggestions or thoughts? As I am at a lost currently.

Any assistance is appreciated.




I do not have experience using this particular plugin… you could try contacting the authors directly? Otherwise - I great plugin for tracking is TrackMate, which is available with Fiji. It is a well-maintained and user-friendly tool and its author (@tinevez) is active on the forum. Perhaps it’s worth taking a look?