FRET results
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hi,
can anyone give some idea that which is the most appropriate laser wavelength for performing acceptor photobleaching method of CFP/YFP FRET. If 488nm laser is used for YFP excitation is there any possibility of loosing FRET data. What difference does it create in FRET results if we use 514nm laser line.
thanks & best wishes,
RK
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Usually we use 514 nm line from the argon laser to bleach or
excite the YFP or Venus. 488nm is close to CFP excitation wavelength and you
may bleach a lot of CFP molecule compared with 514nm. Ammasi Periasamy, Ph.D. Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), McCormick Rd University of Virginia Charlottesville, VA 22904 Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210; Email:[hidden email] http//:www.kcci.virginia.edu ************************ Workshop on FRET Microscopy, March 9-13, 2010 http://www.kcci.virginia.edu/workshop/workshop2010/index.php ************************* From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of ramesh
kandpal hi, can anyone give some idea that which is the most appropriate
laser wavelength for performing acceptor photobleaching method of CFP/YFP FRET.
If 488nm laser is used for YFP excitation is there any possibility of loosing
FRET data. What difference does it create in FRET results if we use 514nm
laser line. thanks & best wishes, RK |
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In reply to this post by ramesh kandpal
Hi,
We use 514 nm for yfp and 405 or 458 for cfp. Please be aware of the potential artifact that comes from bleaching ypf and then exciting cfp with 405:
We do get a very strong (artificial) signal in the donor channel when using 405 nm even in a control prep where there is no donor at all! It seems to be a conversion of the yfp molecules to a form that can be easily excited by 405, but not nearly as much by 458. There was a paper about this around 2000 in Nature, I think. Some authors say they don't see this conversion, but we do. Our recipe now is to excite cfp with 458. I hope this helps,
Zoltan
On Fri, Apr 3, 2009 at 12:55 PM, ramesh kandpal <[hidden email]> wrote:
-- Zoltan Cseresnyes Facility manager, Imaging Suite University of Cambridge, UK |
 
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Alberto Diaspro |
Biophysics in Genoa -Summer 2009
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In reply to this post by Periasamy, Ammasi (ap3t)
Yep we didn’t use the 488 line
either, always the 514nm [the argon 514nm line is spot on for the YFP peak
anyway]. Not sure is we even tried the 488nm line as it’s clearly not
optimal for YFP [expect we did as it was there, and rapidly rejected it]. The *488nm
line hits the tip of the CFP excitation peak [~4% excitation] and misses the
YFP peak [excitation down from 100% at 514nm to 41% at 488nm], and so it is well
worth avoiding the 488nm line and going for 514nm line for YFP if you can. I know we did try the violet 405nm diode laser
for CFP and it didn’t work as well as the ~450nm argon laser line [in
fact the 405nm line was rather poor]. We were using a Leica SP2 with the
optional FRET module, which made it all rather easier when FRET imaging. Most
problems were getting FRET consistency between samples [worked well one month
and no so good the next] rather than poor CFP/YFP FRET as such. Regards Keith *I assume you know the excellent Invitrogen
‘spectral viewer’ for looking at fluorophore spectral
excitation/emissions: --------------------------------------------------------------------------- From:
Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t) Usually we use 514
nm line from the argon laser to bleach or excite the YFP or Venus. 488nm is
close to CFP excitation wavelength and you may bleach a lot of CFP molecule
compared with 514nm. Ammasi Periasamy, Ph.D. Director, Keck Center for Cellular Imaging (KCCI) Professor of Biology and Biomedical Engineering Biology, Gilmer Hall (064), McCormick Rd University of Virginia Charlottesville, VA 22904 Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210; Email:[hidden email] http//:www.kcci.virginia.edu ************************ Workshop on FRET Microscopy, March 9-13, 2010 http://www.kcci.virginia.edu/workshop/workshop2010/index.php ************************* From:
Confocal Microscopy List [mailto:[hidden email]] On Behalf Of ramesh kandpal hi, can anyone give some idea that which is the most
appropriate laser wavelength for performing acceptor photobleaching method of
CFP/YFP FRET. If 488nm laser is used for YFP excitation is there any
possibility of loosing FRET data. What difference does it create in FRET
results if we use 514nm laser line. thanks & best wishes, RK |
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