Posted by
Rajendran, Megha on
URL: http://imagej.273.s1.nabble.com/Flat-field-correction-of-fluorescent-images-tp5000936.html
Hi,
We use epifluorescence microscopy to study FRET using lanthanide metals.
We are just venturing into quantitative analysis and I am having some
issues correcting my images for uneven illumination.
(The image is of the metal solution in capillary for calibration and the
flat-field image was captured by putting a drop of lanthanide on a slide.)
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.
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.
(I also tried the correction using the original image, by blurring it and
then using it as flat-field image. I still see the high values. sometimes
the max values are as high as 60000.)
Let me know if you need more information
Thanks,
Megha
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