Confocal Microscopy List

Re: FRET results

Posted by Keith Morris on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FRET-results-tp2580297p2592377.html

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:

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.reg.uk.html

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Dr Keith J. Morris,
Molecular Cytogenetics and Microscopy Core,
Laboratory 00/069 and 00/070,
The Wellcome Trust Centre for Human Genetics,
Roosevelt Drive,
Oxford OX3 7BN,
United Kingdom.

Telephone: +44 (0)1865 287568
Email: [hidden email]
Web-pages: http://www.well.ox.ac.uk/cytogenetics/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Periasamy, Ammasi (ap3t)
Sent: 03 April 2009 14:38
To: [hidden email]
Subject: Re: FRET results

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

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of ramesh kandpal
Sent: Friday, April 03, 2009 7:56 AM
To: [hidden email]
Subject: FRET results

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,