1. Victor Sourjik ZMBH, University of Heidelberg
EMBO Practical course on Quantitative FRET, FRAP and FCS Live-cell FRET
Victor Sourjik
ZMBH, University of Heidelberg

2. Choose fluorescent labels
Measuring FRET in vivo
Define the goal
Choose fluorescent labels
Choose your method
Get data!

3. I. Goals of in vivo FRET measurements
Measuring molecular distances
Detecting conformational changes
Detecting interactions
Localizing interactions
Following interaction dynamics
Reporting enzymatic activities and intracellular conditions

4. Measuring molecular distances using FRET
High efficiency
FRET efficiency is very sensitive to the distance between fluorophores
 potential of FRET as a molecular ruler
FRET efficiency for CFP/YFP FRET pair
FRET Efficiency:
E = R06/(R06+R6)  1/R6
No FRET at R > 11 nm (100 Å)
GFP size ~ 5 nm (50 Å) R R0
R06  J*QD*n-4*2
Low efficiency

5. Measuring molecular distances using FRET
FRET efficiency is very sensitive to the distance between fluorophores
 potential of FRET as a molecular ruler
Problems of in vivo FRET
Fluorophores are usually large (fluorescent proteins) and coupled with flexible linkers
Limited attachment sites for fluorophores
Weak specific fluorescence (due low to moderate protein levels)
High autofluorescence background
Non-opimal ratio of donor to acceptor

6. Measuring molecular distances using FRET
FRET efficiency is very sensitive to the distance between fluorophores
 potential of FRET as a molecular ruler
Problems of in vivo FRET
Fluorophores are usually large (fluorescent proteins) and coupled with flexible linkers
Limited attachment sites for fluorophores
Weak specific fluorescence (due low to moderate protein levels)
High autofluorescence background
Non-opimal ratio of donor to acceptor
Possible (although not ideal) solution:
Fix the cells and use fluorescently-labeled monoclonal antibodies

7. Measuring molecular distances using FRET
FRET efficiency is very sensitive to the distance between fluorophores
 potential of FRET as a molecular ruler
Problems of in vivo FRET
Fluorophores are usually large (fluorescent proteins) and coupled with flexible linkers
Limited attachment sites for fluorophores
Weak specific fluorescence (due low to moderate protein levels)
High autofluorescence background
Non-opimal ratio of donor to acceptor
Ideal solution:
Labeling with small dyes

8. Detecting conformational changes using FRETP
High efficiency
Low efficiency

9. Detecting conformational changes using FRET
Advantages
Ratio of donor to acceptor is fixed P
Problems
Precision is frequently not high enough (general for measuring distances)
Limited attachment sites for fluorophores

10. Detecting conformational changes using FRET
Advantages
Ratio of donor to acceptor is fixed P
Problems
Precision is frequently not high enough (general for measuring distances)
Limited attachment sites for fluorophores
Most common current uses:
Conformational changes in complexes
Reporter of intracellular conditions

11. Detecting conformational changes in complexes
Advantages
Conformational changes are typically larger
Problems
Ratio of donor to acceptor is not fixed P P

12. Detecting conformational changes in complexes
Advantages
Conformational changes are typically larger
Problems
Ratio of donor to acceptor is not fixed
Possible solution:
Use only one fluorophore (homo-FRET) P P

13. FRET as reporter of intracellular conditions
Advantages
Sensors are engineered to exhibit large conformational changes upon ligand binding or modification
CaM
Problems
Only a limited number of sensors is available: Ca2+, cAMP, several kinases...
Ca2+
CaM
Based on conformational chenge, e.g. Cameleon (calcium sensor)

14. FRET as reporter of intracellular conditions
Advantages
Sensors are engineered to exhibit large conformational changes upon ligand binding or modification
Binding domain
Phosphorylation domain
Problems
Only a limited number of sensors is available: Ca2+, cAMP, several kinases... P
Based on intramolecular binding, e.g. kinase reporters

15. Detecting protein interactions using FRET
Interacting proteins (or, more exactly, proteins in one complex)
Promises
FRET as a generalized interaction- mapping technique
Problems
Strong spectral cross-talk between typical fluorophores (fluorescent proteins)
Typically low FRET efficiency
Limited attachment sites for fluorophores
Weak specific fluorescence
Non-opimal ratio of donor to acceptor
Bulky fluorophores
 Detection of absolute strength of physiological interactions is non-trivial
Non-interacting proteins

16. Detecting protein interactions using FRET
+ Stimulus
Possible solution:
Detecting changes in protein interactions
Relative concentrations of donor and acceptor do not change upon stimulation (i.e., internal control)
 Changes in FRET are more reliably detected than absolute values P
- Stimulus

17. II. Fluorescent labels for in vivo FRET measurements
Fluorescent proteins
In-vivo labeling with fluorescent dyes

18. Proteins vs dyes in fluorescence microscopy
Fluorescent proteins
Can be genetically encoded (high specificity)
Proteins are bulky (5 nm)
Spectra are broad (strong cross-talk)
Not very bright and photostable
In-vivo labeling with fluorescent dyes
Small size
Bright and relatively photostable
Narrow spectra and large spectral choice
Specific in-vivo labeling is difficult

19. Spectral requirements for FRET labels
CFP = cyan fluorescent protein (donor)
YFP = yellow fluorescent protein (acceptor)
Requirements for the FRET pair:
excitation spectra of donor and acceptor are separated
emission spectrum of donor overlaps with excitation spectrum of acceptor
emission spectra of donor and acceptor are separated

20. Fluorescent proteins for in vivo FRET measurements
Nathan C. Shaner, Paul A. Steinbach, & Roger Y. Tsien Nature Methods, Vol. 2: 905 – 909
Any two proteins with overlapping emission spectrum of donor and excitation spectrum of acceptor can be used a FRET pair (including the same protein as donor and acceptor)

21. Fluorescent proteins for in vivo FRET measurements
Caution: FRET efficiency with FPs as FRET pair is always far below 100%

22. Fluorescent dyes for in vivo FRET measurements
Fluorescent dyes with relatively specific binding to short peptide sequences (e.g., FlAsH or ReAsH)
Miyawaki et al., supplement to Nature Cell Biol., 5
Fluorescent dyes specifically binding to protein tags (e.g., SNAP-tag or HaloTag)
HaloTag, Promega Corporation

23. Combining proteins and dyes for in vivo FRET measurements
Roger Y. Tsien’s web site

24. III. Methods to measure FRET in vivo
Spectral measurements
Two-channel FRET (sensitized emission)
One-channel FRET (acceptor photobleaching)
One-channel FRET (donor photobleaching)
Polarization imaging
Life-time imaging

25. Spectral measurement of FRET
Advantages
Complete spectral information
Drawbacks
Requires a specialized system (e.g., Zeiss LSM 710)
Requires carefull image analysis

26. Spectral measurement of FRET

27. Spectral measurement of FRET
In a general case (so-called linear spectral unmixing):
Acquire spectra at donor and acceptor excitation wavelength
Acquire spectra for control samples with only donor and only acceptor
Subtract donor and acceptor cross-talk (bleed-through) to get true FRET signal

28. Two-channel measurement of FRET
Advantages
Can be performed on a simple wide-field microscope
Drawbacks
Limited spectral information
Requires carefull image analysis

29. Two-channel measurement of FRET Sensitized emission A B C
Linear spectral unmixing
Leica Microsystems

30. One-channel measurement of FRET Acceptor photobleaching
Procedure:
Acquire signal of donor fluorescence
Bleach acceptor
Acquire signal of donor fluorescence again
510 nm

31. One-channel measurement of FRET Acceptor photobleaching
Advantages
Is very simple and reliable
Drawbacks
One-time experiment
510 nm

32. One-channel measurement of FRET Acceptor photobleaching
Imaging
Whole-field acquisition
YFP
CFP
510 nm
Can be done either in imaging or whole-field acquisition mode

33. One-channel measurement of FRET Donor photobleaching
Donor (CFP) fluorescence
+ FRET
- FRET
Time (sec)
Advantages
Is comparatively simple
Drawbacks
One-time experiment
Can be affected by other intracellular factors
Procedure:
Follow kinetics of donor bleaching

34. Polarization (anisotropy) measurement of FRET
Weak (no) FRET = high anisotropy
Strong FRET = low anisotropy
Homo-FRET
Advantages
Allows measuring homo-FRET
Is comparatively simple
Drawbacks
Requires specialized equipment
Can be affected by other intracellular factors
Procedure:
Excite with polarized light
Measure emission in two orthogonal directions of polarization

35. Life-time measurement of FRET
Time (sec) ps fs ns
Phizicky et al., Nature :208-15

36. Life-time measurement of FRET
Time (sec)
Advantages
Reports both FRET efficiency and fraction of interacting proteins
Not sensitive to acceptor concentration
Drawbacks
Limited speed
Limited spatial resolution
Phizicky et al., Nature :208-15

37. Our own work (just one slide!) FRET as a network mapping technique
Bacterial chemotaxis network A B