At 11:19 AM 3/27/01 +0200, you wrote:
>    We are trying to do FRET with CFP and YFP, which are the most common
>combination used for this purpose. We have Chroma filters: Ex 436/20 BS
>455DCLP and Em 480/40 for CFP; Ex 500/20 BS Q515LP and Em 520LP for YFP;
>and Ex 436/20 BS 455DCLP and Em 520LP for FRET.
>    I have several questions:
>    1) Do you think this is the best combination? What about GFP and
>DsRed?
>    2) We have found a lot of bleedthrough of CFP in FRET filter set.
>Does it usual? How do you solve it? Do you use other emission filter for
>FRET and YFP?
>    3) If it is not possible to avoid it, which is the kind of image
>calculations do you usually develop? I suppose that it is very important
>to have similar levels of signal with both constructions and set the
>same exposure times when acquiring pictures in a CCD camera, isn't it?
>Thank you very much in advance. I look forward to reading your comments.


Carlos,
        In looking over your filter sets specs they will work but you may be
better off to rethink them.  I would be happy to send you the information
on the sets that are used in our optical imaging facility at UTHSCSA.
Several investigators using our facility have been successful in
calculating FRET for various combinations of fluorescent proteins (B/G,
C/Y, and C/DsR) as well as fl/rh on antibodies.  With the help of Chroma
we've tried to optimize our filter sets to reduce bleedthrough but nothing
is perfect. It is therefore essential to make corrections.  Various
corrections schemes have been published but we continue to use the 3
specimen/ 3 filter set scheme that was originally published by Gordon et
al. (1998)  Biophysical Journal 74:  2702-2713.  We've placed these
algorithms in an Excel spreadsheet and have our acquisition/analysis
software (Metamorph) set up to log the intensity data to the spreadsheet
directly.  Contact me if you would like specifics.
        You may be interested in attending our upcoming symposium on
"FRET and FLIM: Advanced Fluorescence Techniques for Biological Imaging"
that will be held in San Antonio June 8-10, 2001
(http://usa.hamamatsu.com/fretflim/).
        Cheers,
                Vickie Frohlich
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Victoria Centonze Frohlich, Ph.D.
Associate Director
Department of Cellular & Structural Biology's
Optical Imaging Facility
Mail Code 7762
University of Texas Health Science Center at San Antonio
7703 Floyd Curl Drive
San Antonio, TX. 78229-3900

Email:  mailto:[log in to unmask]
Phone:  210-567-3151
Fax:    210-567-3803
http://www.uthscsa.edu/csb/csbhome.html


FRET/FLIM Symposium June 8-10, 2001
http://usa.hamamatsu.com/fretflim/


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