fret texx\aching module

8/9/2019 FRET TeXX\aching Module

1/34

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/


2/34

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

S. Terjung + T. Zimmermann

EAMNET FRET teaching module


3/34

The resolving power of light microscopes is limited to distances of

hundreds of nanometers (


4/34

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.




5/34

Dependence on distance and spectral overlap



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.


6/34

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)




7/34

FRET detection methods

A variety of FRET detection methods exist for light microscopy

Acceptor photobleaching

Donor photobleaching

Ratio imaging

Sensitized emission

Fluorescence lifetime measurements




8/34

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




9/34

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



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


10/34

Acceptor photobleachingShift Correction by Cross-Correlation helps avoiding edge artifacts in the comparison ofpre- and postbleach images.

Edge

artifacts



Without correction With correction


11/34


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




12/34

Overview1) Fluorescence Resonance Energy Transfer Basics

2) Confocal FRET detection techniques

3) FRET and fluorescent proteins





13/34

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)




14/34

GFP-Protein GFP-Protein

P

d>Ro

d


15/34

Acceptor photobleachingReceptor phosphorylation after EGF-Stimulation

0 min 2 min 5 min

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



CFP/YFP


16/34

CFP/YFPThe combination of cyan and yellow fluorescent protein is the

most commonly used fluorescent protein FRET pair




17/34

Fluorescence Resonance Energy Transfer Cameleon Tandem constructs

CFP YFP

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



In vivo CFP/YFP cameleon measurements


18/34

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

2 µM Ionomycin+ 20mM CaCl2

Histamine EGTA




19/34

Sensitized emission detection


20/34


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



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



21/34


Predetermined factors with pure samples of donor and acceptor:Donor cross-talk : RD Acceptor cross-excitation: RE



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

=>


22/34

Overview1) Fluorescence Resonance Energy Transfer Basics

2) Confocal FRET detection techniques

3) FRET and fluorescent proteins




CFP/YFP


23/34

CFP/YFPCyan and yellow fluorescent protein is the most commonly used

fluorescent protein FRET pair




24/34

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


25/34

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




26/34

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 Pairs


27/34

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



Comparison of CFP/YFP and


28/34

Comparison of CFP/YFP and

GFP2/YFP FRET pairs

CFP YFP

exc. 405/458 nm

glycine linker

GFP2 YFP

exc. 405 nm

glycine linker




29/34

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


30/34

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


31/34

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.



Comparison of CFP/YFP and GFP2/YFP


32/34

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




33/34

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/



Lit t


34/34

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



fret texx\aching module

Documents