To normalize Pixel intensities allowing for differing pixel/micron ratios

[Trevor Grigg]

How can you normalize the mean gray value of pixel intensities 8  bit (0-255)taken of cells when the images are at different magnifications 400x or 630x  with pixels/micron being at 3.723 for some images and 5.882 for other images. I am measuring the mean pixel intensity of each cell (ROI) and the distribution of these means I am using to form a box and whisker chart to measure the fluorescence of my peptide within the population of cells. I am new to using Image J but I’m on a fast learning curve and I’m asking if someone can please assist me. Thank you

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[Herbie]

This is a cross-post from the ImageJ-Forum:
<https://forum.image.sc/t/normalizing-pixel-areas-in-microns-of-different-magnification-images-taken-on-a-leica-confocal/20788>

Good day Trevor,

I'd recommend to measure the same object with both microscope settings.
The reason is that the optics will have different transfer characteristics.

Regards

Herbie

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Am 10.11.18 um 17:56 schrieb Trevor Grigg:
> How can you normalize the mean gray value of pixel intensities 8  bit (0-255)taken of cells when the images are at different magnifications 400x or 630x  with pixels/micron being at 3.723 for some images and 5.882 for other images. I am measuring the mean pixel intensity of each cell (ROI) and the distribution of these means I am using to form a box and whisker chart to measure the fluorescence of my peptide within the population of cells. I am new to using Image J but I’m on a fast learning curve and I’m asking if someone can please assist me. Thank you
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

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