I am hard-pressing to find a procedure that successfully finds all nuclei given that they are overlapping and of widely varying intensity. But I’m attcahing a pipeline that gives it a shot. My thought process is the following:
- Illumination correction to remove the high flourescenet back within the nuclei mass. For some reason, using the Regular/division method didn’t work, so I used the Background/subtraction approach to bring the intensity of the nuclei within the cluster down to the same level as those outside.
- Smoothing the image helps with the removing the dark dimples within each nuclei. I assume this is what you were attempting the feature suppression? If that’s the case, I think your feature size was too big, on the order of the nuclei, rather than the size of the dimples which are throwing off segmentation. It also gives the same net effect as the FFT approach in ImageJ (which I couldn’t get working on my computer). Some further adjustment here might be necessary.
- Thresholding by intensity seems to give somewhat better results, although ordinarily I’d prefer Laplacian of Gaussian for cases like this. Also i think the correction factor needs some adjustment, I’m just not sure by how much.
Hope this helps!
2011_09_09.cp (5.11 KB)