Re: Flat-field correction of fluorescent images
Posted by
pang on
URL: http://imagej.273.s1.nabble.com/Flat-field-correction-of-fluorescent-images-tp5000936p5000963.html
Dear Megha,
I don't think that your dark current noise is 1044.771. It should be slight greater than your minimum intensity (501). I had published a paper and wonder if it can give you some information you need.
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2011.03581.x/abstractZhengyu
Zhengyu Pang, Ph.D.
Biochemistry and Bioanalytics Laboratory
Diagnostic and Biomedical Technologies
GE Global Research, K1-5B37A
One Research Circle
Niskayuna, NY 12309
T: 518-387-4015
F: 518-387-7765
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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