Re: Quantifying overlap on fluorescence image
Posted by
John Hayes on
Apr 13, 2019; 3:14am
URL: http://imagej.273.s1.nabble.com/Quantifying-overlap-on-fluorescence-image-tp5021990p5022073.html
I hate to be a “Debbie Downer” (
https://en.wikipedia.org/wiki/Debbie_Downer); but again Rohitesh, I wonder what you/your adviser/your supervisor are getting at here. You certainly should split the channels, but if I have to (partly) get “nerdy/technical” here, I’m wondering how cleanly you expect these channels to be? They are not as clean as you may have been led to believe in a microscope training session as I was (GFP, just 500-530 nm — that’s what I was taught! — WRONG). Again, NOT QUANTITATIVE!! There’s inevitably going to be SOME overlap between the RGB channels… The degree of overlap, and the importance thereof, is subjective in my view — that’s where to attack in a referee report. If you start exactly spouting percentages of overlap from these data, for the reviewer, even better to shoot you down!
If you look at the fluorescence spectra of common fluorophores found here:
http://www.spectra.arizona.edu/ and here:
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.htmlThey are NEVER cleanly separated across spectra of fluorophores like software RGB cleanly are.
I hate to be the only one that craps on this for you (and I’m the only one on the mailing list that thinks this?), and maybe I’m being nitpicky, but I DO wish you all the best.
Again, I wish you the best; but I thought I’d add just my 2c because I’m trying to giving you CONSTRUCTIVE advice and not just INSTRUCTIVE advice…
Cheers,
John
> On Apr 12, 2019, at 9:30 PM, Rohitesh Gupta <
[hidden email]> wrote:
>
> I want to ask this again if anyone has had success with coloc2 ? I am trying diff ways to rectify my images but I still get that conventional warning message. I am just thinking of splitting the images and measuring fluorescence independently for each channel. It doesn't seem like that coloc2 is indeed a good option.
>
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