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

On Mon, 6 Oct 2003 18:58:16 -0400
  Jennifer Genova <[log in to unmask]> wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I know I love getting 4 or 5 of the same email, but just
>in case you skipped
>over it.  Keep fingers crossed for FRET!
>
>Jenn
>
>On 10/6/03 2:47 PM, "Scott Snyder" <[log in to unmask]>
>wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Dear list,
>>       Something our lab has been considering is to look
>>at FRET between an XFP
>> nuclear receptor and a DNA binding dye.  Molecular
>>probes sells a wide
>> variety of these so it looks like there should be
>>several possible
>> donor/acceptor combinations.  The thing that makes us
>>nervous is that it
>> just seems too easy.  Someone should have published on
>>it by now if it were
>> possible but I see no literature references in a search
>>of PubMed.  Has
>> anyone tried something like this?  Is there some problem
>>we are missing?  I
>> thought about the possibility that a noncovalent
>>interaction between DNA dye
>> and DNAwould throw things off but most of these
>>interactions are stable
>> enough for preloading on electrophoresis (thus should be
>>fairly long lived).
>>       Along a similar note, has anyone ever determined a
>>Forster radius for
>> CFP/YFP or any of the other XFP FRET pairs?  I keep
>>hearing numbers quoted
>> but have never been able to find a source.
----
Hi Scott

FRET appears to be easy but there are number of issues
involved in FRET imaging. Depending on your experimental
condition you can optimize your fluorophore selection and
labeling. Also its important to do the control experiment.
If you need more about it you can email me off the list or
attend our upcoming workshop on FRET Microscopy (March
10-13, 2004). The workshop announcement will appear in the
list in a week or two.

We covered some of the issues involved in FRET imaging in
the following papers listed below for various
applications. you can also see many papers in the
literature related to FRET to verify whether your approach
is published or not.

(1) Wallrabe, H., Elangovan, M., Burchard, A., Periasamy,
A. and Barroso, M. (2003) Confocal FRET microscopy to
measure clustering of receptor-ligand complexes in
endocytic membranes. Biophysical J. 85:559-571.
(2) Mills, J.D., Stone, J.R., Rubin, J.D., Melon, D.E.,
Okonkwo, D.O., Periasamy, A. and Helm, G.A. (2003)
Illuminating Protein Interactions in Tissue using Confocal
and Two-photon Excitation Fluorescent Resonance Energy
Transfer (FRET) Microscopy. J. Biomed. Opt. Vol.8(3),
347-356.
(3) Sekar, R.B. and Periasamy, A. (2003)  Fluorescence
resonance energy transfer (FRET)  microscopy imaging of
live cell protein localization. J. Cell Biol.,
160:629-633.
(4) Day, R.N., Voss, T.C., Enwright III, J.F., Booker,
C.F., Periasamy, A. and Schaufels, F. (2003) Imaging the
localized protein interactions between Pit-1 and the
CCAAT/enhancer binding protein alpha (C/EBPá) in the
living pituitary cell nucleus. Mol. Endo. 17(3), 333-345.
(5) Elangovan, M., Wallrabe, H., Chen, Y., Day, R.N.,
Barroso, M. and Periasamy, A. (2003) Characterization of
one- and two-photon excitation resonance energy transfer
microscopy. Methods. 29, 58-73.
(6) Elangovan, M., Day, R.N. and Periasamy, A. (2002) A
novel nanaosecond FRET-FLIM microscopy to quantitate the
protein interactions in a single living cell. J
Microscopy. 1:2-14.



Ammasi Periasamy, Ph.D.
Director, Keck Center for Cellular Imaging (KCCI)
Professor of Biology and Biomedical Engineering
Gilmer Hall (064), McCormick Rd
University of Virginia
Charlottesville, VA 22932
Voice: 434-243-7602 (O); 982-4869 (lab)
Fax:434-982-5210; Email:[log in to unmask]
http//:www.kcci.virginia.edu
************************
"Methods in Cellular Imaging" Oxford University Press.
Edited by A. Periasamy; Covers topics on Confocal,
Multiphoton, FRET, FLIM, TIRF, and other advanced
techniques and various biological applications.
*************************
attend the International Conference on Multiphoton
Microscopy in the Biomedical Sciences. Visit the web site
http://www.spie.org/Conferences/
*************************