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FRET Basics and Applicationsan EAMNET teaching module
Timo Zimmermann + Stefan TerjungAdvanced Light Microscopy Facility
European Molecular Biology Laboratory, Heidelberg
http://www.embl.de/almf/http://www.embl.de/eamnet/
Overview
1) Fluorescence Resonance Energy Transfer Basics
2) Confocal FRET detection techniques
3) FRET and fluorescent proteins
4) A new GFP FRET pair with increased efficiency
S. Terjung + T. ZimmermannEAMNET FRET teaching module
The resolving power of light microscopes is limited to distances of hundreds of nanometers (<organelles).Fluorescence Resonance Energy Transfer (FRET) allows the detectionof molecule-molecule interactions in the nanometer range with light microscopy.
FRET is sometimes also called Förster Resonance Energy Transfer, as Förster was the first who published quantitative theory of molecularresonance energy transfer (Förster 1946, Förster 1948).
1 nm10-9m
1 µm10-6m
1 mm10-3m
1 cm10-2m
1 m1 Å10-10m
Cells Worm HouseflyOrganelles Human
FRET
light microscopyresolution limit
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Fluorescence Resonance Energy transfer (FRET)
FRET is a non-radiative transfer of energy from an excited donormolecule 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 resultsnot only in donor emission, but partially also in emissioncharacteristic for the acceptor fluorophore.
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Dependence on distance and spectral overlap
S. Terjung + T. ZimmermannEAMNET FRET teaching module
The efficiency of energy transfer strongly depends on the distance between the donoracceptor molecules and on overlap of the donor molecule emission and acceptor moleculeexcitation spectra high specificity.
FRET efficiency is dependson molecule distance
and
The FRET efficiency depends on the distance between the two interacting molecules. At the distance of the Förster radius R0 between the molecules, the FRET efficiency is 50%. The typical R0 is around 3 nm.
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)
S. Terjung + T. ZimmermannEAMNET FRET teaching module
FRET detection methods
A variety of FRET detection methods exist for light microscopy
Acceptor photobleaching
Donor photobleaching
Ratio imaging
Sensitized emission
Fluorescence lifetime measurements
S. Terjung + T. ZimmermannEAMNET FRET teaching module
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
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Acceptor PhotobleachingExperimental steps of acceptor photobleaching measurementsIn acceptor photobleaching, the acceptor molecule of the FRET pair is bleached, resulting in a brightening (unquenching) of the donor fluorescence.
PrebleachImage Bleaching Postbleach
ImageMedian Filtering
Subtraction: Postbleach –
Prebleach
Division: Subtraction/ Postbleach
Zoom4x
OriginalZoom
GFP GFP
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Cy3 Cy3
488 488 488
543 543543
An apparent FRET efficiency (productof the efficiency of the FRET pair and the amount of interacting donor) canbe calculated
Acquisition Processing
Acceptor photobleachingShift Correction by Cross-Correlation helps avoiding edge artifacts in the comparison of pre- and postbleach images.
Edge artifacts
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Without correction With correction
Donor photobleaching
FRET decreases donor fluorescence lifetime=> decreased likeliness of bleaching=> decreased bleaching rate
Fluorescencelifetime
The bleaching rate of the donor fluorophore is affected by FRET. Measuring the bleaching of the donor in the presence/absence of acceptor is a possibility to detect FRET.
An apparent FRET efficiency (product of the efficiencyof the FRET pair and the amount of interacting donor) can be calculated.However: Quantitation is problematic due to direct and indirect bleaching of acceptor
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Overview
1) Fluorescence Resonance Energy Transfer Basics
2) Confocal FRET detection techniques
3) FRET and fluorescent proteins
4) A new GFP FRET pair with increased efficiency
S. Terjung + T. ZimmermannEAMNET FRET teaching module
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-ProteinP
d>Ro
d<Ro
Measurement of Protein Phosphorylation by FRET
Cy3 anti-P-Thyr
Application example: An acceptor-labelled antibody against a phosphorylatedresidue can be used to detect the phosphorylation status of a GFP-fusionprotein by FRET
PhosphorylatedNot Phosphorylated
Verveer, et al. 2000
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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/YFPThe combination of cyan and yellow fluorescent protein is themost commonly used fluorescent protein FRET pair
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Fluorescence Resonance Energy TransferCameleon Tandem constructs
CFP YFP
Pollock and Heim TiCB 1999, Miyawaki et al. Nature 1997
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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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Cross-talk and cross-excitation of two fluorophores is an intrinsic problem of multichannelmeasurements and is also present in FRET measurements
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Channel 1:460-500 nm
Channel 2:460-500 nm
RGBOverlay
CFPonly
YFPonly
CFP+YFP
Sensitized emission detection
DA
D
A
DA
D
A
Ratiometric imaging canonly be done in sampleswith a fixed stochiometryof donor and acceptor(e.g. Cameleons)
D
A
A
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DA In samples with variable
stochiometries, the detectedacceptor fluorescence has to becorrected for emission cross-talkand for cross-excitation
ADA
AD
A D AA
Sensitized emission detectionPredetermined factors with pure samples of donor and acceptor:
Donor cross-talk : RDAcceptor cross-excitation: RE
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Donor channelDonor excitation
FD
Acceptor channelDonor excitation
FDA
Acceptor channelAcceptor excitation
FA
corr
Donorcross-talkcorrection
Acceptorcross-excitation
correction
Required images:
FDA corr/FA
=>
Overview
1) Fluorescence Resonance Energy Transfer Basics
2) Confocal FRET detection techniques
3) FRET and fluorescent proteins
4) A new GFP FRET pair with increased efficiency
S. Terjung + T. ZimmermannEAMNET FRET teaching module
CFP/YFPCyan and yellow fluorescent protein is the most commonly usedfluorescent protein FRET pair
S. Terjung + T. ZimmermannEAMNET FRET teaching module
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
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Spectral overlap of FRET Pairs
The spectral overlap of donor emission and acceptor excitation isonly partial for CFP/YFP and much better for GFP/YFP pairs
S. Terjung + T. ZimmermannEAMNET FRET teaching module
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
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Different Cross-Excitation of FRET PairsUsing a suitable laser excitation for CFP, YFP is directly excitedsignificantly (=> 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 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
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Improved FRET Efficiency significantly improves Detection
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.
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Sensitized emission of GFP2-YFP FRET pairs
GFP2excitation
GFP2emission
GFP2excitation
YFPemission
YFPexcitation
YFPemission
YFP (sensitized emission)YFP (direct excitation)
GFP2+YFPCoexpression
GFP2-YFPlinked
Data are shown after linear unmixing of the GFP2 and YFP emission signals.
S. Terjung + T. ZimmermannEAMNET FRET teaching module
Comparison of CFP/YFP and GFP2/YFP FRET pairs
- 32% increased overlap of donor emission and acceptorexcitation
- 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
S. Terjung + T. ZimmermannEAMNET FRET teaching module
ALMF: Rainer PepperkokJens RietdorfStefan 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/
S. Terjung + T. ZimmermannEAMNET FRET teaching module
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 Letters
531, 245 -249
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