two color live cell imaging- ratio data

Posted by Knecht, David on
URL: http://imagej.273.s1.nabble.com/two-color-live-cell-imaging-ratio-data-tp3702347.html

We are trying to sort out a localization issue for one of our GFP  
probes.  The localization is relatively subtle relative to  
cytoplasmic background so we want to be sure it is correct since  
unfused probes are not uniform either.  We have co-expressed our GFp-
fusion and mRFP (cytoplasmic probe) in the same cells to address this  
question.
1.  WHat is the best way to handle this data?  It seems this should  
be obvious, but I am struggling with it. FIrst choice is to simply  
make a red-green or cyan-magenta merge, but that requires balancing  
of the two channels and I think it is easy to get fooled trying to  
look for color differences. Second, one could background subtract  
(green-red) but that requires a careful balancing of the two channel  
intensities plus may generate negative numbers.  Alternatively, we  
could do a ratio (green/red), but that seems to ask a slightly  
different question. Ratio seems right to me as a way of highlighting  
the relative difference in localization between the two probes.
2.  Assuming one does a ratio (I am using Ratio Plus), the numbers  
come out in the 0-5 range as 32 bit data. How is the result handled  
in ImageJ to generate the kind of color image that I am used to  
seeing from ratio data.  Is it a special LUT or do you mathematically  
bring the numbers to a 255 scale and then apply a normal LUT.
Thanks- Dave

Dr. David Knecht
Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)