Noise or error? The effect of cells with weak movement (**) on output kinematic parameter!

[yousof mohammadi]

Hello everyone,

I’m using the OpenCASA plugin (Motility module) to compute VCL, VAP, and VSL on human semen samples. My settings are:

Microns/pixel: 0.97

Min cell size (µm²): 1

Max cell size (µm²): 100

STR% (progressive): 80

VAP> (progressive): 25

Minimum VCL (µm/s): 10

VCL lower threshold (µm/s): 60

VCL upper threshold (µm/s): 150

Frame rate (fps): 60

Minimum track length (frames): 30

Maximum displacement between frames (µm): 8

Window size (frames): 4

Imaging details: Basler aca800-510uc camera on an inverted microscope (10× objective). I recorded ~2 seconds as .mp4, then converted it to black-and-white .avi in MATLAB before running OpenCASA.

I’m separating highly motile sperm from raw samples using a microfluidic device. The key point is that the microfluidic fraction contains only actively motile sperm, whereas the raw (unprocessed) sample is a mixture of cells with both high and very low motility.

My question concerns the cells with very weak movement (the head shows slight oscillation in place; the cells are alive but essentially near-zero speed). OpenCASA marks these tracks with **. When I compare the raw sample to the microfluidic fraction, the plugin excludes these ** cells from the calculations, which artificially inflates the raw sample’s average VCL,VAP,VSL.

How can I adjust the settings so that these near-stationary (**) cells are still tracked and included in the output statistics (Motility, VCL, VAP, VSL), to get a more accurate comparison between raw and microfluidic groups?

Additional note: In the raw sample video, the cells marked with ** are clearly visible. Currently, only tracks that carry a label are included in the VCL/VAP/VSL calculations, which should not be the case—the ** tracks should also be tracked and included in the velocity metrics.

Thanks

[f.martin...@gmail.com]

Hi Yosuof,

If you have no drift or immotile spermatozoa being bumped by motile ones, just try by reducing the "Minimum VCL" setting. Anything below that speed is considered immotile. Setting to 0 should capture anything moving minimally (if they displace frame-to-frame, I don't think it works if they only vibrate on place).

Good luck!

Felipe

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