Hello world .
I’d like to ask for some help on how to count nuclear speckles (gamma-H2A.X foci)
I have worked with the Speckle counting example pipeline and made it work, but I’m not satisfied with the segmentation of the foci.
Images were taken with a Leica TCS SP5 confocal microscope and a 63x/1.4 oil immersion objective
The problem is that a number of cells have a rather diffuse staining where the foci are not so well defined. The pipeline as it is now (attached below) in some cells detects speckles which I would say are not existing and on the other hand fails to detect some that are clearly present (pics of example cells are attached, white arrows highlight false positives, yellow arrows point to false negatives).
I’ve played around with the IdentifyPrimaryObjects module for the speckles:
Increasing object diameter didn’t help
I’ve increased the threshold correction factor, which helped a bit, but with further increase I get more false negatives
For detection of clumped objects I tried settings Intensity and Laplacian of Gaussian and the latter seems to work better
I didn’t manage to optimize the segmentation by adjusting the smoothing filter size and maxima suppression distance
Any help regarding which parameters could be changed in which way to improve foci detection would be greatly appreciated.
Some original full-size images to work with I’ve also attached as “Input images.zip” (Hoechst DNA stain in the blue channel has ch01 in the file name, the green channel with the gamma-H2A.X signal has ch00 in the filename).
Thank you very much in advance
Input Images.zip (962 KB)
ds_gH2AX-foci_use me.cp (14.1 KB)