Gert-Jan Kremers FRET and Live Cell Imaging Wednesday, May 21,
QFM 2014
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FRET microscopy Outline 1.What is FRET and what is it used for?
2.Requirements for FRET? 3.How is FRET measured? 4.FRET in the real
world
Slide 3
Fluorescence microscopy can be used to determine if two
proteins are co-localized. This means that they are found within
the same ~250 nm x ~500 nm region of the specimen. However,
proteins are much smaller than the resolution limit of
fluorescence. Is there any way we can use fluorescence microscopy
to detect where specific protein-protein interactions occur?
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Yes, using FRET!
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What is FRET? Frster (fluorescence) resonance energy transfer,
or FRET, is a distance-dependent, photophysical process between a
donor and acceptor fluorophore FRET results in a loss of donor
fluorescence and an increase in donor fluorescence FRET occurs
between fluorophores separated by distances of less than 10nm FRET
can easily be measured by fluorescence microscopy FRET can thus be
used to report on the proximity of molecules over much smaller
distances than can be resolved by standard fluorescence or even
super-resolution approaches
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What is FRET used for? Protein-protein interaction
(intermolecular FRET) Conformational change (intramolecular
FRET)
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What is FRET used for? http://zeiss.magnet.fsu.edu/ Ca 2 +
Genetically encoded biosensors
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Example: Androgen receptor Ligand-dependent transcription
factor t = 45min Martin van Royen, Erasmus MC
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Androgen receptor DBDNTDYFPCFPLBD DBDNTDYFP CFP LBD
DBDNTDYFPLBD DBDNTDYFPLBD DBDNTDCFPLBD DBDNTD CFP LBD Both intra-
and intermolecular N/C interaction Intermolecular N/C interaction
only Questions: 1.Do androgen receptors dimerize? (Intermolecular
N-C interactions) 2.Does ligand binding induce conformational
change? (Intramolecular N-C interaction) 3.In what order do these
events occur? DBDNTDLBD DBDNTDLBD DBDNTDLBD DBDNTDLBD NTD DBDLBD
DBDLBD
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Schaufele, et al. (2005) PNAS Van Royen et al. (2007) JCB
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What are the requirements for FRET to occur? 1.Spectral overlap
between donor emission and acceptor absorbance spectra 2.Favorable
orientation of fluorophores 3.Close proximity (less than 10nm)
4.Frster radius (R 0 )
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1.Spectral overlap between donor emission and acceptor
absorbance spectra Add legend to graph
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2. Favorable orientation Vogel et al 2006
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3. Close proximity of donor and acceptor Vogel et al 2006
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3. Close proximity of donor and acceptor E = 1 / [1 + (r/R o )
6 ]
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4. Frster radius (R 0 ) R 0 = [2.8x10 -11 2 QY D A J()] 1/6 nm
2 = Orientation factor, range 0 to 4, but 2/3 for randomly oriented
D and A. QY D = donor quantum yield A = acceptor extinction
coefficient J()= spectral overlap integral = F D () E A () 4 d
Slide 17
R 0 for common FRET pairs DonorAcceptorR 0 (nm) FITCTMR5.5
Cy3Cy55.0 ECFPEYFP4.7 EGFPmCherry5.3 mTurq2mVenus5.8
mClovermRuby26.3
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FRET microscopy Outline 1.What is FRET and what is it used for?
2.Requirements for FRET? 3.How is FRET measured? 4.FRET in the real
world
Sensitized acceptor fluorescence Excite donor, measure acceptor
fluorescence +True readout of FRET +Easy qualitative measurements
Quantitation requires many correction for bleedthrough, etc.
Sensitive to photobleaching Vogel et al 2006
Slide 22
FRET with CFP and YFP Excitation of CFP leads to some YFP
excitation because YFP is brighter than CFP. CFP emission also
bleeds into the YFP channel (i.e. there will always be some
apparent FRET signal).
Wavelength excitation FluorophoreDonor emission images Acceptor
emission images D Dab Ac D + Aef A Ad g Need 3 filter sets and 3
cell preparations D, donor; A, acceptor D, donor excitation
wavelength; A, acceptor excitation wavelength Calculating
Bleedthrough Factors Precision FRET (PFRET) Algorithm
Slide 25
PFRET = UFRET DSBT ASBT UFRET (image f) is uncorrected FRET
ASBT is the acceptor spectral bleedthrough ASBT = ([c]/[d]) x [g].
DSBT is the donor spectral bleedthrough DSBT = ([b]/[a]) x [e].
!Assumes the images of single and double-labeled samples are
collected under identical conditions. !Assumes bleedthrough is
proportional to the amount of donor and acceptor present in a given
cell.
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Spectral imaging Excite donor, measure emission spectra
+Spectral information +Easy qualitative measurement Sensitive to
photobleaching Low S/N ratio FRET No FRET
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Fluorescence lifetime imaging +Concentration independent
+Acceptor does not need to be fluorescent +Quantify fraction of
interacting molecules Special (expensive) instrumentation Low
number of photons
Slide 28
Polarization anisotropy imaging
http://www.microscopyu.com/articles/fluorescence/fret/fretintro.html
+Fast +Nondestructive +Single sample +Can also measure homoFRET
Difficult with high NA lenses
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Acceptor photobleaching / donor dequenching Bleach acceptor
Donor quenched due to FRET Release of donor quenching
+Straightforward and quantitative: E = (I D - I DA )/I D +Performed
on single sample Destructive Beware of artifacts due to acceptor
photoconversion upon bleaching
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Quantifying FRET using acceptor photobleaching Post bleach Pre
bleach E = (I D - I DA )/I D E = (63 - 42)/ 63 = 0.34 34% FRET E =
(I D - I DA )/I D E = (94 - 61)/ 94 = 0.35
Slide 31
FRET microscopy Outline 1.What is FRET and what is it used for?
2.Requirements for FRET? 3.How is FRET measured? 4.FRET in the real
world (the good, the bad and the ugly)
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Piston and Kremers (2007) TIBS 32:407
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If FRET is so great, why isnt everyone doing it? FRET analysis
requires rigorous controls and careful quantification Acceptor is
often excited directly Spectral overlap Not all molecules within
FRET proximity undergo FRET (orientation effects) Not all
interacting proteins will show FRET FRET is not immune to
experimental artifacts
Slide 34
Special considerations for FRET studies using fluorescent
proteins Remember that FRET measures the distance between the
fluorophores, which in this case are buried inside the beta barrel
of the FPs Many fluorescent proteins themselves can form dimers or
higher-level oligomers when present at high concentrations, and
this can give rise to a false positive See Zacharias et al (2002)
Science 296:913 Thus is important to use monomeric forms for FRET
studies
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Biophys J. 2006 December 15; 91(12): L99L101 A good way to get
started: FRET standards as a positive control
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Another type of positive control: known interacting proteins
LC3 and Atg4B C74A have been reported to directly interact Kraft
and Kenworthy 2012 J Biomed Optics
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Slide 38
Slide 39
Nature Methods 2, 801 (2005) doi:10.1038/nmeth1105-801 Valentin
et al. Photoconversion of YFP into a CFP-like species during
acceptor photobleaching FRET experiments Example of a FRET
artifact
Slide 40
Examples of ways to demonstrate FRET is specific Include
negative controls matched for expression level and localization
Demonstrate FRET is eliminated when interacting domains are mutated
Show physiological regulation of FRET Unlabeled proteins should
compete with labeled proteins
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