Plotting spectra and estimating spillovers...

Adrian Smith A.Smith at centenary.usyd.edu.AU
Fri Jul 22 06:57:10 EST 2005


Dear all,

I've been asked to prepare some plots for our users showing the 
emission and excitation spectra of various fluorochromes.

A simple enough request, especially considering the proliferation of 
spectral viewing tools (eg BDs, Molecular Probes, Frank Battye's etc) 
and the availability of many spectra as txt files.

However, I also been asked to scale the spectra to reflect the 
relative intensties ("brightness") of each flurochrome and make any 
other adjustments necessary to estimate the amount of compensation 
between the flurochromes when using different fluorochrome 
combinations and/or filters.

Now, to my understanding, IF I could obtain some measure of relative 
"brightness" I MIGHT be able to scale the peaks to give the viewer 
some idea of relative intensity (given the same target of course). 
The question is where would I find such data in a form that I could 
use? And even if I could find such "brightness" data what about other 
variables like differential responses to laser power etc?

As for estimating spillover and compensation, it would seem to me 
that the number of variables involved would be too large to be 
meaningfully accounted for on simple diagram? Or maybe the there is 
one dominant variable that might allow a plot to be drawn that would 
allow a rough estimation?

Has anyone ever attempted such a project? If so, did you have any success?

Are there any more confounding factors that I've overlooked? (ie that 
I could add to my list of why spending a long time on this be would a 
waste of time and why it would be better to spend the time on 
physical experiments on our machines to produce some data specific to 
our situation).


Regards,

Adrian Smith
Centenary Institute, Sydney, Australia.



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