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Hi, S.P. - 

Are you saying that you see increase in both cfp & yfp signal independent of
your bleaching step? Do you see this in all your samples including controls?
Are you monitoring your laser power? You can do this with the monitor diode
if you have one on your system or you can use the transmitted light
detector. I would check that the laser power is stable over the course of
your experiment. Have you tried not fixing? I.e., doing live-cell
experiments? Are you sure your CFP is pure? Sometimes it can be contaminated
with YFP. What happens if you use GFP and RFP? 

-Holly
__________________
Holly L. Aaron
CRL Molecular Imaging Center
http://imaging.berkeley.edu

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Stephen Whitefield
Sent: Friday, March 02, 2007 8:16 AM
To: [log in to unmask]
Subject: Increase after YFP bleaching in CFP/YFP FRET

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Dear Confocal List,

We are having a problem with FRET analysis on a Zeiss LSM 510 META. We are 
using HEK 293 cells transfected with CFP-YFP positive controls and co-
transfected Protein A -YFP and Protein B -CFP and fixed in 3.5% PFA/ 10 
mins. We have tried both Vectashield, Prolong and Sigma mounting media. We 
are encountering an increase in both CFP and YFP intensity after photo 
bleaching YFP outside the bleach area. The increase in intensity is 
comparable to the increase we see in CFP in the ROI area after acceptor 
photo bleaching which makes FRET analysis difficult. We would appreciate 
any comments that might explain what is going on and if there are possible 
remedies for this problem.
Thank you

S. P. Yegorova
Dalhousie University
Pharmacology Department
(902)494-2651
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