Image Analysis, count the number of red and green cells, and the number of overlaps

Python
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Hello, I have many nd2 files brain (nikon microscope files that can be converted to tif/jpeg) and need to count the number of red cells, green cells, and their overlap in a given region of interest (coordinates of rectangle found visually/pulled from an excel file containing known locations of this brain structure).

For example, I’ve attached one of these images with the green and red channels split:

M3_8_A_Whole_10x_g1_resized
M3_8_A_Whole_10x_g1_resized1606×1307 390 KB

I would like to count the number of green and red dots in the yellow rectangle shown in the green channel and see the overlap of these cells.

p.s. I had to resize the JPEG from 17839 X 14517 to 30% of orginal dimensions in order to upload here.

Thank you,

Timothy Tax

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M3_8_A_Whole_10x_r1_resized
M3_8_A_Whole_10x_r1_resized1920×1563 23.2 KB

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What research have you done into your assignment? What approach(es) have you attempted?

5

I’ve attempted to use ImageJ, but the automatic thresholding algorithms are not accurate in every image. I’ve attempted using Imaris as well, which works fine, but each image takes about 5 minutes to count the cells and I have hundreds and a two week deadline haha.

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