Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

We find that both CFP and YFP, but especially CFP, undergo a short-term
(minutes) partial recovery from the photobleaching that inevitably
occurs when you are setting up the specimen due to the casual exposure
to UV or laser illumination.  One strategy is to wait 5 minutes before
starting the acceptor photobleach.  Another is to subtract off the
recovery using an unexposed area of similar brightness as a control. 

Richard Hallworth, Ph.D., Associate Professor
Director, Nebraska Center for Cell Biology
http://www.biomedsci.creighton.edu/facilities/nccb.html
Department of Biomedical Sciences
Creighton University School of Medicine
2500 California Plaza
Omaha NE 68178
Ph: (402) 280-3057
FAX: (402) 280-2690
Email: [log in to unmask]

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Holly Aaron
Sent: Tuesday, March 06, 2007 2:08 PM
To: [log in to unmask]
Subject: Re: Increase after YFP bleaching in CFP/YFP FRET

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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
[log in to unmask]