Posted by
Daniel James White on
Jun 03, 2010; 8:57am
URL: http://imagej.273.s1.nabble.com/Re-Colocalization-analysis-w-ICA-and-JACoP-No-intensity-saturation-please-tp3688067.html
Hi Bryan,
Looking at cell1_Green.tiff and the red one that goers with it,
it is clear that the images are very badly over exposed / clipped / saturated.
in cell2 the green is not saturated, but there is a saturated pixel in the red channel.
(its significant as the image is small)
cell3 is badly saturated.
This is very very bad news in general for quantitative analysis and especially for coloc analysis.
Yuo cant use images like this in this kinf of analysis.
You will get false/meaningless results.
Essentially, you do not know the true intensity of the brightest pixels,
since their intensities were chpoosed off at the top of the detectors range.
You lost the info that you are most interested in.
You must collect image data with in the range of the detectors ability.
You must use a HiLo or similar range indicator look up table / pallette
to set the imaging parameters of the detector so the background is just above zero,
and the brightest pixels are not at the top of the range (typically 255 or 4095 etc. )
When I see no saturated pixels - i really mean NONE.
Also the offset is set a bit high: there should be very few or no 0 pixels,
to make sure you dont lost the info at the lowe end of the scale also.
You also need good signal:noise for these method,
as the methods used to measure coloc are very sensitive to noise.
For a tutorial on how to get it right look here:
http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysissorry for the bad news,
but I prefer to point you in the right direction than let you get it wrong.
I don't mean to cause embarrassment.
By no means are you the only person who ever made these mistakes, nearly everyone does.
cheers
Dan
On Jun 3, 2010, at 6:00 AM, IMAGEJ automatic digest system wrote:
Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany
+49 (0)15114966933 (German Mobile)
+49 (0)351 210 2627 (Work phone at MPI-CBG)
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http://www.chalkie.org.uk Dan's Homepages
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dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )