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Zhan,
Autofluorescence can exist in fret-related complexes.  Lipofuscin in human
brain tissue is an example (see reference below).  So if the autofluorescent
acceptor is susceptible to bleaching when you bleach your target acceptor,
it will contribute to the overall fret.  You might be able to characterise
the level of contribution by autofluorescence fret on each measurement using
spectral analysis if the autofluorescence is part of a longer chain (as it
is in the case of lipofuscin). Do this by looking for changes in
fluorescence at wavelengths higher then your acceptor emission.
Alternatively the autofluorescence might have a different lifetime.
Otherwise you only be able to correct for an average amount of
autofluorescence fret.

Hashemzadeh-Bonehi L, Phillips RG, Cairns NJ, et al.
Pin1 protein associates with neuronal lipofuscin: Potential consequences in
age-related neurodegeneration
EXPERIMENTAL NEUROLOGY 199 (2): 328-338 JUN 2006

Hope this helps,
Roger

Dr Roger Guy Phillips
Centre for Advanced Microscopy,
University of Sussex
School of Life Sciences
John Maynard Smith Building
Falmer, Brighton & Hove
BN1 9QG
United Kingdom

phone:44 (0)1273 877585
fax: 44 (0)1273 678433
email: [log in to unmask]
room:2C9 (ext 7585)/lab 4C2 (ext 2734)

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]On
Behalf Of zhan Tom
Sent: 19 September 2006 02:40
To: [log in to unmask]
Subject: how to solve autofluorence in FRET


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hello everyone,
    I met a strange problem when I try to do FRET(CFP-YFP)experiment with
the protoplast of arabidopsis.I try to bleach YFP using 515nm laser, and it
works. after bleaching,the autofluorence seemed brighter than pre-bleach.I
can see the CFP channel become brighter, but the autofluorence of the
chloroplast also become brighter.So I worry about the FRET result will be
affected by autofluorence.
   has anyone met the similar problem? any reply about autofluorence of
plant is appreciated.
   sincerely,
    zhan cheng

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