Re: Flat-field correction of fluorescent images

Posted by Nico Stuurman on
URL: http://imagej.273.s1.nabble.com/Flat-field-correction-of-fluorescent-images-tp5000936p5000937.html

Hi Megha,

On Nov 22, 2012, at 10:27 AM, Rajendran, Megha wrote:

> I have a set of flat-field images at different gain. I was trying to correct one image using imageJ as follows:
> 1. converted both my images to 32 bit (from 16/12 bit).
> 2. subtracted both images with average dark current noise image.
> 2. normalized my flatfield image by dividing with the max signal.

I think that you will want to normalize your flatfield image with the average (not max) signal.  That should also  lower the values after flatfielding.

> 3. divided my original image with the normalized flatfield image.
> When I check the plot profile across the diagonal, the field is even. Then i measure the mean, min and max gray value. I compared this with the
> values before correction.
>                   Area           Mean            Min     Max
> Before flat-field: 1310720 1337.228 699.752 1900.651
> After flat-field:  1310720 1899.424 661.128 8839.022
>
> I don't know why the values are so high after correction. I was wondering
> if I am missing a step here.

Best,

Nico

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