Problem with automated cell counting for DAB stained Fos+ cells in brain slides

Posted by Praz Nina on May 26, 2014; 10:11am
URL: http://imagej.273.s1.nabble.com/Problem-with-automated-cell-counting-for-DAB-stained-Fos-cells-in-brain-slides-tp5007921.html

Hi everyone,

I'm trying to devise a method to enable me to do automated cell counting in DAB stained Fos+ cells in brain slides, but I'm having trouble because the count varies from the manual count. I have tried many different options, but essentially I first sharpen the original image (16bit, but greyscale), subtract the background (radius 10) and then convert to 8-bit and do thresholding. Then I analyze particles (size 50-Infinity, circularity 0.56-1.00) and sometimes have an ok match, but most of the times not really.

The issue is that I'm not sure (even in manual counting) if I'm counting only the stained cells or if I'm including also the ones from the layer below, since my slides look like 4096 shades of grey :).

Here is a sample image: https://i.imgur.com/m9uDJBN.jpg

(I know the image is a bit out of focus, an undergrad took them so they're not exactly tack sharp..)

How would you set the threshold, which cells would you count? I find it strange that I can "see better" which cells to count when I zoom out to like 12.5%, but when I'm at 100%, it become ambiguous. This image has been taken with a 10x lens btw, but I also have a stitched panorama taken with a 20x lens. And although the number of pixels is much higher, it doesn't really help, as the background is equally as distracting.

Any assistance will be greatly appreciated!