fret texx\aching module
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FRET Basics and Applicationsan EAMNET teaching module
Timo Zimmermann + Stefan Terjung Advanced Light Microscopy Facility
European Molecular Biology Laboratory, Heidelberg
http://www.embl.de/almf/http://www.embl.de/eamnet/
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Overview
1) Fluorescence Resonance Energy Transfer Basics
2) Confocal FRET detection techniques3) FRET and fluorescent proteins
4) A new GFP FRET pair with increased efficiency
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The resolving power of light microscopes is limited to distances of
hundreds of nanometers (
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Fluorescence Resonance Energy transfer (FRET)
FRET is a non-radiative transfer of energy from an excited donor molecule to a suitable acceptor molecule in close proximity.
Wouters et al. (2001), TICB 11/5
Fluorescence Resonance Energy Transfer
In the case of FRET, excitation of the donor fluorophore results
not only in donor emission, but partially also in emission
characteristic for the acceptor fluorophore.
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Dependence on distance and spectral overlap
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The efficiency of energy transfer strongly depends on the distance between the donor
acceptor molecules and on overlap of the donor molecule emission and acceptor moleculeexcitation spectra high specificity.
FRET efficiency is depends
on molecule distance
and
The FRET efficiency depends on the distance between the two interacting molecules. Atthe distance of the Förster radius R0 between the molecules, the FRET efficiency is 50%.
The typical R0 is around 3 nm.
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Donor/Acceptor Pairs
Examples for common FRET Donor/Acceptor pairs:
Donor (Em.) Acceptor (Exc.)
FITC (520 nm) TRITC (550 nm)
Cy3 (566 nm) Cy5 (649 nm)
EGFP(508 nm) Cy3 (554 nm)
CFP (477 nm) YFP (514 nm)
EGFP (508 nm) YFP (514 nm)
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FRET detection methods
A variety of FRET detection methods exist for light microscopy
Acceptor photobleaching
Donor photobleaching
Ratio imaging
Sensitized emission
Fluorescence lifetime measurements
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FRET detection methods
The detection methods have different properties and are suitedto different samples
Detection of changes:
Acceptor photobleaching
Donor photobleaching
Information self-contained:
Ratio imaging
Sensitized emission
=> fixed samples
=> in vivo
Fluorescence lifetime measurements
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Acceptor Photobleaching
Experimental steps of acceptor photobleaching measurements
In acceptor photobleaching, the acceptor molecule of the FRET pair is bleached,resulting in a brightening (unquenching) of the donor fluorescence.
Prebleach
ImageBleaching
Postbleach
Image
Median
Filtering
Subtraction:
Postbleach –
Prebleach
Division:
Subtraction/
Postbleach
Zoom
4x
Original
Zoom
GFP GFP
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Cy3 Cy3
488 488 488
543 543543
An apparent FRET efficiency (productof the efficiency of the FRET pair andthe amount of interacting donor) canbe calculated
Acquisition Processing
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Acceptor photobleachingShift Correction by Cross-Correlation helps avoiding edge artifacts in the comparison ofpre- and postbleach images.
Edge
artifacts
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Without correction With correction
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Donor photobleaching
FRET decreases donor fluorescence lifetime=> decreased likeliness of bleaching=> decreased bleaching rate
Fluorescence
lifetime
The bleaching rate of the donor fluorophore is affected by FRET.
Measuring the bleaching of the donor in the presence/absence ofacceptor is a possibility to detect FRET.
An apparent FRET efficiency (product of the efficiency
of the FRET pair and the amount of interacting donor)
can be calculated.
However: Quantitation is problematic due to direct and
indirect bleaching of acceptor
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Overview1) Fluorescence Resonance Energy Transfer Basics
2) Confocal FRET detection techniques
3) FRET and fluorescent proteins
4) A new GFP FRET pair with increased efficiency
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FRET and Fluorescent Proteins (FPs)
Protein-Protein Interactions:
- FRET between an FP and a dye
- FRET between FPs
Cameleons:
In vivo measurements of physiologicalchanges (ratio imaging)
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GFP-Protein GFP-Protein
P
d>Ro
d
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Acceptor photobleachingReceptor phosphorylation after EGF-Stimulation
0 min 2 min 5 min
ErbB1-GFP/Cy3 FRET (receptor phosphorylation), Verveer, et al. 2000
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CFP/YFP
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CFP/YFPThe combination of cyan and yellow fluorescent protein is the
most commonly used fluorescent protein FRET pair
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Fluorescence Resonance Energy Transfer Cameleon Tandem constructs
CFP YFP
Pollock and Heim TiCB 1999, Miyawaki et al. Nature 1997
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In vivo CFP/YFP cameleon measurements
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In vivo CFP/YFP cameleon measurementsMeasurements caried out on the Leica SP2 AOBS at 405 nm excitation:
2 µM Ionomycin+ 20mM CaCl2
Histamine EGTA
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Sensitized emission detection
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Sensitized emission detection
D
A
D
A
D
A
D
A
Ratiometric imaging can
only be done in samples
with a fixed stochiometryof donor and acceptor
(e.g. Cameleons)
D
A
A
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DA In samples with variable
stochiometries, the detected
acceptor fluorescence has to becorrected for emission cross-talk
and for cross-excitation
A
DA
A
DA
DA
A
Sensitized emission detection
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Sensitized emission detection
Predetermined factors with pure samples of donor and acceptor:Donor cross-talk : RD Acceptor cross-excitation: RE
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Donor channel
Donor excitation
FD
Acceptor channel
Donor excitation
FDA
Acceptor channel
Acceptor excitation
FA
corr
Donor
cross-talk
correction
Acceptor
cross-excitation
correction
Required images:
FDA corr/FA
=>
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Overview1) Fluorescence Resonance Energy Transfer Basics
2) Confocal FRET detection techniques
3) FRET and fluorescent proteins
4) A new GFP FRET pair with increased efficiency
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CFP/YFP
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CFP/YFPCyan and yellow fluorescent protein is the most commonly used
fluorescent protein FRET pair
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Requirements for a good FRET pair
-Maximal overlap of donor emission and
acceptor excitation-Minimal direct excitation of the acceptor at theexcitation maximum of the donor
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Spectral overlap of FRET Pairs
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Spectral overlap of FRET Pairs
The spectral overlap of donor emission and acceptor excitation is
only partial for CFP/YFP and much better for GFP/YFP pairs
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Requirements for a good FRET pair
-Maximal overlap of donor emission and
acceptor excitation-Minimal direct excitation of the acceptor at theexcitation maximum of the donor
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Different Cross-Excitation of FRET Pairs
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Different Cross Excitation of FRET Pairs
Using a suitable laser excitation for CFP, YFP is directly excited
significantly (=> high background signal)GFP2 is excitable around 400 nm, where YFP is almost not excitable
(=> low background signal)
458 405
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Comparison of CFP/YFP and
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Comparison of CFP/YFP and
GFP2/YFP FRET pairs
CFP YFP
exc. 405/458 nm
glycine linker
GFP2 YFP
exc. 405 nm
glycine linker
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Acceptor photobleachingComparison of CFP and GFP2 in the same construct
Before After
CFP-YFP: FRET efficiency 20%
GFP2-YFP: FRET efficiency 30%
=> 50% increase
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Improved FRET Efficiency significantly improves Detection
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Whereas the differences between FRET pairs are not significant at high
transfer efficiencies, a more efficient FRET pair significantly improvesthe detectable FRET interaction in cases of low FRET efficiency.
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Sensitized emission of GFP2-YFP FRET pairs
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p
GFP2
excitation
GFP2
emission
GFP2
excitation
YFP
emission
YFP
excitation
YFP
emission
YFP (sensitized emission)
YFP (direct excitation)
GFP2+YFP
Coexpression
GFP2-YFP
linked
Data are shown after linear unmixing of the GFP2 and YFP emission signals.
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Comparison of CFP/YFP and GFP2/YFP
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FRET pairs
- 32% increased overlap of donor emission and acceptor excitation
- Higher absorbance and quantum efficiency of the donor
- Higher Foerster Radius (approx. 5.5 nm)
- Increased FRET efficiency, especially at longer distances- Suitable for donor photobleaching
- However: Linear unmixing of the strongly overlappingemission signals required
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ALMF: Rainer PepperkokJens Rietdorf
Stefan Terjung
GFP2/YFP project: Andreas GirodVirginie Georget
Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2 -YFP FRET pair
T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, R. Pepperkok, FEBS Letters 531 (2002)245 -249
http://www.embl.de/almf/http://www.embl.de/eamnet/
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Lit t
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Literature• T. Förster (1946): Naturwissenschaften 6, 166
• T. Förster (1948): Ann. Phys. (Leipzig) 2, 55
• A. Miyawaki, J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura and R. Y.
Tsien (1997): Nature 388, 882-887.
• B.A. Pollok and R. Heim (1999): Trends in Cell Biology 9, 57-60.
• P.J. Verveer, F.S. Wouters, A.R. Reynolds, P.I. Bastiaens (2000): Science 290, 1567-
1570
• F.S. Wouters, P. J. Verveer and P. I. H. Bastiaens (2001): Trends Cell Biol 11, 203-211.
• T. Zimmermann, J. Rietdorf, A. Girod, V. Georget, R. Pepperkok (2002): FEBS Letters531, 245 -249
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