Re: 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-tp3702347p3702350.html

A follow up question to my question.  If I do 32 bit math and divide  
one channel by another or by a constant, I get pixel values ranging  
from roughly 0-2 (with three decimal significance).  If I then apply  
the r-g ratio LUT through the Image/color/edit LUT, it applies the  
LUT as if the data is 0-255.  Is it autoscaling the LUT to the  
dataset?  Is there some way of setting the scale and/or determining  
what values were assigned what colors?  I would like different images  
to have the same scaled LUT, but they won't if it is autoscaled.  
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)


On Jun 26, 2006, at 10:09 AM, David Knecht wrote:

> 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)