Posted by
carlo bianco on
URL: http://imagej.273.s1.nabble.com/Flat-field-correction-of-fluorescent-images-tp5000936p5000964.html
hello,
I'd like to know if is possible to set a 100 point grid
for point counting, I've got the grid plugin, but it sets
the grid with areas, not with number of points.
Thank you in advance for your help
bye
carlo bianco
bologna university
Italy
________________________________
Da: "Pang, Zhengyu (GE Global Research)" <
[hidden email]>
A:
[hidden email]
Inviato: Lunedì 26 Novembre 2012 21:35
Oggetto: Re: Flat-field correction of fluorescent images
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/abstract
Zhengyu
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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