Posted by
Jacqueline Ross on
URL: http://imagej.273.s1.nabble.com/Flat-field-correction-of-fluorescent-images-tp5000936p5000968.html
Hi Carlo,
You could try modifying the DrawRandomDots.txt macro if you want random placement of the points (
http://rsbweb.nih.gov/ij/macros/DrawRandomDots.txt).
It puts 25 random dots on an image but you could change that to 100 and modify the size of the dots so that they aren't too large depending on your image resolution..
Kind regards,
Jacqui
Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND
Tel: 64 9 923 7438
Fax: 64 9 373 7484
http://www.fmhs.auckland.ac.nz/sms/biru/-----Original Message-----
From: ImageJ Interest Group [mailto:
[hidden email]] On Behalf Of carlo bianco
Sent: Tuesday, 27 November 2012 9:57 a.m.
To:
[hidden email]
Subject: 100 point grid for point counting
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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