Timo Zimmermann + Stefan Terjung Advanced Light · PDF fileTimo Zimmermann + Stefan Terjung...

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


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.


Dependence on distance and spectral overlap


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)


FRET detection methods

A variety of FRET detection methods exist for light microscopy

Acceptor photobleaching

Donor photobleaching

Ratio imaging

Sensitized emission

Fluorescence lifetime measurements


FRET detection methods

The detection methods have different properties and are suitedto different samples

Detection of changes:

Acceptor photobleaching


Information self-contained:

Ratio imaging

Sensitized emission

=> fixed samples

=> in vivo

Fluorescence lifetime measurements


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


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


Without correction With correction


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


Overview






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)


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


Acceptor photobleachingReceptor phosphorylation after EGF-Stimulation

0 min 2 min 5 min

ErbB1-GFP/Cy3 FRET (receptor phosphorylation), Verveer, et al. 2000


CFP/YFPThe combination of cyan and yellow fluorescent protein is themost commonly used fluorescent protein FRET pair


Fluorescence Resonance Energy TransferCameleon Tandem constructs

CFP YFP

Pollock and Heim TiCB 1999, Miyawaki et al. Nature 1997


In vivo CFP/YFP cameleon measurementsMeasurements caried out on the Leica SP2 AOBS at 405 nm excitation:

2 µM Ionomycin+ 20mM CaCl2

Histamine EGTA


Cross-talk and cross-excitation of two fluorophores is an intrinsic problem of multichannelmeasurements and is also present in FRET measurements


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


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


Donor channelDonor excitation

FD

Acceptor channelDonor excitation

FDA

Acceptor channelAcceptor excitation

FA

corr

Donorcross-talkcorrection

Acceptorcross-excitation

correction

Required images:

FDA corr/FA

=>

Overview






CFP/YFPCyan and yellow fluorescent protein is the most commonly usedfluorescent protein FRET pair


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


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


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


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


Comparison of CFP/YFP and GFP2/YFP FRET pairs

CFP YFP

exc. 405/458 nm

glycine linker

GFP2 YFP

exc. 405 nm

glycine linker


Acceptor photobleachingComparison of CFP and GFP2 in the same construct

Before After

CFP-YFP: FRET efficiency 20%GFP2-YFP: FRET efficiency 30%=> 50% increase


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.


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.


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


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/


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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