Fluorescence Resonance Energy Transfer () Microscopy Mike Lorenz Optical Technology Development mlorenz@mpi-cbg.de -FLM course, May 2009
What is fluorescence? Stoke s shift Fluorescence light is always redshifted!!! 1.0 0.8 fluorescence rel. intensity 0.6 0.4 absorption 0.2 0.0 450 500 550 600 650 wavelength / nm Quantum yield Ratio of emitted to absorbed photons Lifetime Average time the fluorophore remains in the excited state
Spectroscopic principles of E = k k + k f + k x = R R 6 0 6 0 + R 6 1,0 R 1948 / 49 2 4 ( Q J n ) 1 κ ( 6 Å 0 = 9790 D λ) n: refraction index Φ D : donor quantum efficiency J: spectral overlap integral κ: dipole orientation factor efficiency E 0,8 0,6 0,4 0,2 0,0 ½ R 0 R 0 1½ R 0 dye-to-dye distance 2 R 0
Förster distance R 0 R ( 2 4 κ Q J ( n ) Å 0 9790 D ) = λ 1 6 κ 2 Q D donor-acceptor orientation factor donor quantum yield J(λ) overlap integral n refraction index 100 ECFP EYFP 80 60 40 20 0 400 450 500 550 600 J wavelength / nm 4 ( λ ) D ( λ) = F ( λ) ε ( λ) 0 dλ D 2 κ = θ θ A T θ D A ( cosθ 3cosθ cosθ ) 2 T D A D A: D A: random: 2 κ 2 κ 2 κ = 4 = 0 = 2 / 3 For most D-A pairs is R 0 = 1.5-7.0 nm
can be measured via ntensity in both channels Fluorescence lifetime of the donor τ τ D DA = k f = k f 1 + k x + k 1 + x k E = 1 D => E τ =1 τ D
Requirements for a good pair Maximal overlap of donor emission and acceptor excitation 100 80 CFP GFP High quantum yield of the donor Good spectral separation norm. intensity 60 40 20 Minimal direct excitation of the acceptor at the excitation maximum of the donor Minimal emission of the donor with the acceptor fluorescence (bleed-through) norm. intensity 0 400 450 500 550 600 650 100 80 60 40 wavelength [nm] CFP YFP 20 0 400 450 500 550 600 650 wavelength [nm]
Common donor/acceptor pairs Donor (Em.) Acceptor (Exc.) R 0 (κ 2 =2/3) FTC (520 nm) TRTC (550 nm) ~ 5 nm Cy3 (566 nm) Cy5 (649 nm) ~ 5.7 nm EGFP (508 nm) Cy3 (554 nm) CFP (477 nm) YFP (514 nm) ~ 5 nm EGFP (508 nm) YFP (514 nm) ~ 5.7 nm EGFP (508 nm) Cherry (588 nm) ~ 5.3 nm
DNA bending measured by
Microscopy R ~ 2-8 n m R > 10 n m!!! CFP ex em YFP ex em 1 kt 0.8 6 ( R0 / R ) = τd optical resolution ~ 200-250 nm λ χ 0.61 NA 0.6 efficiency 0.4 0.2 350 400 450 500 550 Wavelength / nm 600 650 R06 kt E= = 6 k D + kt R0 + R 6
Applications of microscopy
detection methods Donor Photobleaching Acceptor Photobleaching => fixed samples Sensitized Emission Ratio maging Fluorescence Lifetime => in vivo Polarization / Anisotropy
by Acceptor Photobleaching Prebleach mage Acceptor Bleaching (RO) Postbleach mage Filtering Bkg correction Adjust PMT variations Calculate 405 or 456 405 or 456 405 or 456 0.3 460-490 efficiency 0 514 514 514 E = 1 prepb D postpb D 520-620 Acquisition Processing
CFP-YFP: An excellent pair for CLSM 100 80 405 458 514 All laser scanning microscopes have the necessary laser lines for CFP and CFP YFP (458 & 514 nm Argon-Laser). YFP 514 nm excites the acceptor only allowing a selective photobleaching norm. intensity 60 40 Tuneable emission filters can collect most of the CFP fluorescnce without any contribution of the acceptor YFP (460 495 nm) 20 0 400 450 500 550 600 650 wavelength [nm] Recommended CLSM: Leica SP2 or SP5 (with AOBS) Olympus FV1000 Zeiss LSM 710
Problems with CFP-YFP in pb?
Can green-red work as a pair? 514 488 543 594 633 100 80 Cherry GFP R 0 ~ 5.3 nm norm. intensity 60 40 20 0 400 450 500 550 600 650 700 100 80 wavelength [nm] Cherry YFP R 0 ~ 5.7 nm norm. intensity 60 40 20 0 400 450 500 550 600 650 700 wavelength [nm]
by Acceptor Photobleaching 1. Take donor & acceptor image 2. partially pb acceptor 3. Take donor image Microscope: Confocal Advantages: Can be used for all experiments Easy quantitative measurements E ~ 0.13 Disadvantages: Destructive Fixed samples only
by Sensitized Emission CFP-PTB PTB nf = DA YFP-Raver1 D DA D Donor Donor A A Acceptor DA Acceptor Ex: CFP YFP CFP Em: CFP YFP YFP corrected 1 0.8 0.6 CFP YFP ex em ex em Microscope: Widefield / Confocal Advantages: Non-destructive => live cell imaging 0.4 0.2 350 400 450 500 550 600 650 Wavelength / nm Disadvantages: Not quantitative Requires correction for bleedthrough etc. Sensitive to photobleaching
by Sensitized Emission nf can be affected by several factors: Donor and acceptor intensity (or concentration) of the pixels efficiency Ratio of complexes to free donor and acceptor => nf should be normalized to be intensity independent Normalization 1. nf Donor 2. Gordon et al. (1998) N = nf Donor Acceptor Normalization by Gordon (2) is not intensity independent 3. Xia & Liu (2001) N = Donor nf Acceptor (1) and (3) are, but only Xia takes both concentrations into account
by Donor-Acceptor Ratio maging ratio = Donor Ch Ch 435nm CRB WH1 V V CA 470nm 530nm
by Donor-Acceptor Ratio maging ratio = Donor Ch Ch 435nm CDC42 CRB WH1 V V C A 470nm
by Donor-Acceptor Ratio maging Microscope: 0.4 0.7 - ratio Widefield / Confocal Lorenz et al., Curr Biol, 2004 Advantages: Non-destructive => live cell imaging Easy qualitative measurements mages can be taken simultaneously Disadvantages: Limited for biosensors
J Cell Sci. 2004 Mar 15;117(Pt 8):1313-8.
by Anisotropy (Homotransfer) rotational correlation time (~18 ns) >> fluorescence lifetime (<3 ns) Emission light is polarized when excited with polarized light!!! r = + 2 Microscope: Widefield / Confocal Emission light is depolarized. Advantages: Non-destructive => live cell imaging mages can be taken simultaneously Useful for dimerization studies (e.g. receptor dimerization) Only 1 construct is necessary Disadvantages:???
- Microscopy Method: Detects proximity between donor and acceptor fluorophores (up to ~2-8nm) Application: Protein-protein interactions ntramolecular conformational changes Biosensors (e.g. Ca 2+, GTPases, kinases activity) Advantages: ncreases spatial resolution of fluorescence microscopy (~ 200-250nm) Limitations: Absence of is not definitive. Due to a long rotational correlation time of GFP (~18ns) no exact distance information can be obtained.
Literature Review Truong & kura, Curr Opin Struct Biol (2001), 573. Vogel et al., Sci STKE (2006). Acceptor Photobleaching Roy et al., Methods Mol Biol (2009), 69. Sensitized Emission / Ratio maging Gordon et al., Biophys J (1998), 2702. Xia & Liu, Biophys J (2001), 2395. Sorkin et al., Curr Biol (2000), 1395. Lorenz et al., Curr Biol (2004), 697.