Re: Flat-field correction of fluorescent images

Posted by Rajendran, Megha on
URL: http://imagej.273.s1.nabble.com/Flat-field-correction-of-fluorescent-images-tp5000936p5000961.html

Thanks Nico and John,

Sorry for the delayed reply, I wanted to try all the suggestions before replying.

Using the mean intensity solved the problem with high values after flatfield
correction. I was still getting very high min and max values, so I tried smoothing
the Flat-field image using guassian blur (radius 20). These together got me some
sensible values. I am getting negative minimum intensity after subtracting dark
current noise, I am not sure if it matter but wanted to know if I can avoid it (I
switch off all the lights in the room to take these images and average by stacking
and average Z-project).

My values are as follows: (mean intensity; minimum; maximum intensity)
raw image: 1336.853; 501; 4092
substract dark current noise: 1044.771, -1835.300, 2681.300
Divide flat-field (normalized by dividing mean intensity): 1045.247; -1789.488;
3014.886
Without guassian blur, the mean intensity remains same but the min and max intensity
goes crazy.

I have another problem, I plot-profile the 2 diagonals before and after flat-field
correction. one diagonal shows the uneven illumination pattern and corrects
significantly after correction. The other diagonal seems fine to start with but gets
uneven after correcting. I guess it is before my reference images are not right for
my sample. I have tried getting reference images twice at different conditions
(using slide/coverslip and in rectangular capillary, my samples are fluorophore
solution in capillaries), but both the flat-field images have same pattern and it is
different from my samples light pattern. I was wondering if I can do
pseudo-correction using the image itself
(http://www.uhnres.utoronto.ca/facilities/wcif/imagej/image_intensity_proce.htm -
section 7.1.2); has anyone done this before?

Hope I am not confusing things up. Thanks for all the help

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