Does the paper mention any spectral unmixing? I know that some software can mathematically separate GFP and YFP, but proving that you've done it correctly would be tough.  A lot of variables on both the technical and algorithmic techniques. 

I've seen spectral unmixing performed to separate GFP-YFP signals, but only when the labels were distinct. I also am interested how one proves GFP-YFP colocalization, since there are people here who would like to do that as well. 

~Gregg

Gregg Sobocinski
Imaging Specialist/Microscopist
University of Michigan, MCDB Dept.
Ann Arbor, Michigan
USA

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Howard Berg
Sent: Wednesday, May 27, 2009 9:15 AM
To: [log in to unmask]
Subject: GFP/YFP colocalization?

Hi all:

A paper for ''The Plant Cell'' (Raffaele et al.) just came online and  
several figures are presented showing colocalization of GFP and YFP- 
tagged proteins.  I am not surprised that they colocalized because I  
expect there would be a severe bleedthrough for these two.  Am I  
missing something here or have they presented an artifact?  Their  
methods state that they used sequential acquisition (on a Leica TCS  
SP2).

Howard



R. Howard Berg, Ph.D.
Director, Integrated Microscopy Facility
Danforth Plant Science Center
975 N. Warson Rd.
St. Louis, MO 63132

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