Looking for some advice about getting any of the ImageJ plugins to play ball for colocalization of largeish z-stacks (~130 slices).
These stacks are images of cells and I want to only colocalize signal within them, ignoring all signal outside.
I’ve done quite a bit of reading on all the methods available, including using rolling ball or local median background subtraction rather than Costes thresholding.
The only method I have gotten to work so far is using Imaris to create a 3D surface around the cell body, then masking the cell so that all pixels outside are 0 and saving the file. I then open that file in Fiji and running the old ‘colocalization threshold’ plugin set to ignore zero-zero pixels. There appears to be no option to ignore zero-zero pixels in any other colocalization plugin.
This seemed to work on most of my images. However, I am now getting very inconsistent thresholding with this plugin. 7/8 images I tried to analyse today were thresholded wrong and had a tM1/tM2 of 1.0. I believe the Costes method used was somehow inaccurate, and fixed in Coloc 2? However, Coloc 2 freezes (or takes many days, I haven’t left it running because it wouldn’t be feasible anyway) when trying to calculate Costes thresholds.
Anyway, how can I calculate a Costes threshold on a z-stack, ignoring zero-zero pixels, then with that threshold generate Mander’s coefficients etc.?
Should I somehow automatically generate an ROI for each slice in the stack (maybe easier because I already have an intensity of 0 outside of the cell) then apply Coloc 2 to the ROI’d stack?
Thank you in advance for your help and your time.