Calculating z-coordinates

Hello there,

I’m new to these forums and this if my first post/question.

I recently started learning ImageJ for my new job and am having issues quantifying a set of z-coordinates. I’m working with .tiff files showing the movement of e-coli cells and I need to track their movements. I can manually track the x and y coordinates but I am having difficulty figuring out how to get the corresponding z values for each vector point using the depth of field. Is there an easier way to go about tracking this or how can I calculate what I need.

I’ve tried using automatic trackers but given the nature of the videos it’s a bit difficult for me to get the software to distinguish between cell and background when doing any sort of filtering or thresholding.

Any help is greatly appreciated!