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Dear Guenter,

it is very difficult to predict/fit the modulation of the spectral signal of
a given fluorophore by your particular detection device. A good estimate can
typically be derived from a reference measurement of the fluorophores on the
same detection device with low 'background' sample, eg. cultured fibroblast
cells. The 'background' estimation is of course best done on your actual
sample.

Hope that helps, 


Guenter Giese wrote:
> 
> 
> how to do spectral unmixing without "clean" reference signals? 
> 
> Unfortunately, I have no region with single fluorescence signal to be used
> as a reference, and it would be impossible or very time-consuming 
> ...to prepare individual reference probes.
> 
> Is there a way to determine, without "clean" reference signals, the matrix
> values to be used with, e.g., the ImageJ Spectral Unmixing plugin for from
> J. Walter, or for general spectral unmixing purposes? 
> 
> ------------------------------------------
> Dr. Guenter Giese
> Light Microscopy Facility Manager
> Dept. of Biomedical Optics
> MPI fuer Medizinische Forschung Jahnstr. 29
> D-69120 Heidelberg, Germany
> Phone (+49) 6221-486-360 (Fax: -325)
> e-mail: [log in to unmask] 
> 
> 

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