Microscope Focus Quality Plugin - meaning of probabilities stack

Hi all,

I am writing a script to automate the identification of a slice most in-focus within a stack of fluorescence microscopy images. Tried several plugins available and got best results with the Microscope Focus Quality plugin.

It generates a PROBABILITIES STACK from which the first slice seems to match well the evaluation of the focus quality that can be overlay on the image of interest. But I don’t seem to find anywhere the meaning of the other slices in the PROBABILITIES STACK. Does anyone know?

Many thanks,

Allright, in their paper, they refer to using 11 discrete levels of defocusing, which matches the 11 slices present in the PROBABILITIES STACK.

Came to understand that each defocusing level was synthetically achieved by means of a PSF and taking 2 micron steps - which is some serious defocusing (!)

Therefore, it seems that for every patch the probability is calculated for each patch to be in focus (first slice) and at 10 other out of focus levels (the remaining 10 slices).

Let me know if you got another interpretation from the paper.