IlluminationCorrection of overexposed Pictures

Have some problems with the forum, posting again:

Hello,

i did a immunofluorescence staining of brain sections with DAPI (in Blue), Neuronal nuclei marker (in green) and microglia surface marker (in red).

And now i only what to count the blue, the red and the green cells.

For DAPI and neurons there are no problems, but with the microglia i have some trouble: microglias marker is a surface marker, thus they have different morphology and they are clumby.
Also the red channel is overexposed in my pictures.

However, i wanted to normolising the illumination in the red channel, but it doesnt works. The illumination is a big problem, without working on it, its hard to identify the cells…
After that, i want count the red cells, which threshold method do i need?
Maybe, could it be helpful, that i first locate the nuclei with the dapi staining with primaryobjectlocation and after that locating the microglia with secondobjectlocation?

I added a example picture and one picture only the red channel splited and convert to grayscale.




In your images, the red channel is saturated, and this is an image artifact that cannot be corrected via illumination correction. Illumination correction deals with uneven lighting or shading across the image; saturation is another thing entirely.

You can certainly identify the individual nuclei with the DAPI channel since the contrast there looks fine, and the same is true with the green neuronal marker. You might be able to mask out the over-exposed regions and collect data from the remaining areas, but any data coming from the red saturated regions should be considered to be unreliable.

Regards,
-Mark