Why the mean gray value in dark area is less than the light area?

Hello,

I need to quantify the brown stained cells in IHC . However, the dark brow area has less mean gray value than the light brown area on the same slide. Is anyone know why?

Thanks

June

Because that is how digital images work: the intensity of a white pixel is 255 (in an 8-bit channel, or for each channel of an RGB image), and black is zero.

Note that in general the intensity of a chromogenic stain is not a reliable measure of product quantity.

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The dark brown shaped area is so obvious darker than the other areas and I can see the difference by eye. I just need to give a bar figure to show this difference in a grant application. if the dark area has less value than the light area, it will be very confusion to the reader.

Can you please make any suggestion?

Thanks

June

dear @juneva,
could you please upload an example image?
thanks,
Emanuele

how to upload the image?

June

Hi June, you can use the upload button image or just drag and drop the file to the text box.

Tif or Png is fine, although there may be an issue with uploading tifs from ImageJ to this forum.

here is the file
thanks for the help
June

This is the control. you can see difference by the eye that the first photo has dark brown area than the second photo.

Thanks for willing to help

June