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

Posted by ERIC on May 26, 2014; 1:28pm
URL: http://imagej.273.s1.nabble.com/Problem-with-automated-cell-counting-for-DAB-stained-Fos-cells-in-brain-slides-tp5007921p5007925.html

Hi Nina,
As you have a large variation of intensity of your nuclei and a quite
homogenous size, it mayhelp to do a frequency filtering.
I transform (eventualy invert) your image in 8bit did an FFT band pass
filter set to 10pixel for larger size and to 4pixel for smaller size
(see FFT filter.png).
I used the find maxima function (set to 120) to count the nuclei (276
were found see Capture.png).
Of course you will not find the same number by eyes !!!!

run("8-bit");
run("Invert");
run("Bandpass Filter...", "filter_large=10 filter_small=4 suppress=None
tolerance=5 autoscale saturate");
run("Find Maxima...");

Eric Denarier
Grenoble Institut des Neurosciences
Inserm U836
Chemin Fortuné Ferrini
38700 La Tronche
France


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http://neurosciences.ujf-grenoble.fr/

Le 26/05/2014 12:11, Praz Nina a écrit :

> 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!
>
>
>
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>
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