No spectral unmixing was done.  Nor was there any presentation of  
control images showing lack of bleedthrough in GFP- or YFP-only cells...

I agree that unmixing works well for these two fluorophores, provided  
an appropriate level of expression for them both is achieved.

Howard



On May 27, 2009, at 9:25 AM, Sobocinski, Gregg wrote:

> 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
>
> ph 314-587-1261    fx 314-587-1361   cell 314-378-2409
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