Fluorescence Resonance Energy Transfer (FRET) Microscopy

Transcription

1 Fluorescence Resonance Energy Transfer () Microscopy Mike Lorenz Optical Technology Development -FLM course, May 2009

2 What is fluorescence? Stoke s shift Fluorescence light is always redshifted!!! fluorescence rel. intensity absorption wavelength / nm Quantum yield Ratio of emitted to absorbed photons Lifetime Average time the fluorophore remains in the excited state

3 Spectroscopic principles of E = k k + k f + k x = R R R 6 1,0 R 1948 / ( 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

4 Förster distance R 0 R ( 2 4 κ Q J ( n ) Å 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 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 = nm

5 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

6 Requirements for a good pair Maximal overlap of donor emission and acceptor excitation CFP GFP High quantum yield of the donor Good spectral separation norm. intensity 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 wavelength [nm] CFP YFP wavelength [nm]

7 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

8 DNA bending measured by

9 Microscopy R ~ 2-8 n m R > 10 n m!!! CFP ex em YFP ex em 1 kt ( R0 / R ) = τd optical resolution ~ nm λ χ 0.61 NA 0.6 efficiency Wavelength / nm R06 kt E= = 6 k D + kt R0 + R 6

10 Applications of microscopy

11 detection methods Donor Photobleaching Acceptor Photobleaching => fixed samples Sensitized Emission Ratio maging Fluorescence Lifetime => in vivo Polarization / Anisotropy

12 by Acceptor Photobleaching Prebleach mage Acceptor Bleaching (RO) Postbleach mage Filtering Bkg correction Adjust PMT variations Calculate 405 or or or efficiency E = 1 prepb D postpb D Acquisition Processing

13 CFP-YFP: An excellent pair for CLSM 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 Tuneable emission filters can collect most of the CFP fluorescnce without any contribution of the acceptor YFP ( nm) wavelength [nm] Recommended CLSM: Leica SP2 or SP5 (with AOBS) Olympus FV1000 Zeiss LSM 710

14 Problems with CFP-YFP in pb?

15 Can green-red work as a pair? Cherry GFP R 0 ~ 5.3 nm norm. intensity wavelength [nm] Cherry YFP R 0 ~ 5.7 nm norm. intensity wavelength [nm]

16 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

17 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 CFP YFP ex em ex em Microscope: Widefield / Confocal Advantages: Non-destructive => live cell imaging Wavelength / nm Disadvantages: Not quantitative Requires correction for bleedthrough etc. Sensitive to photobleaching

18 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

19 by Donor-Acceptor Ratio maging ratio = Donor Ch Ch 435nm CRB WH1 V V CA 470nm 530nm

20 by Donor-Acceptor Ratio maging ratio = Donor Ch Ch 435nm CDC42 CRB WH1 V V C A 470nm

21 by Donor-Acceptor Ratio maging Microscope: 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

22 J Cell Sci Mar 15;117(Pt 8):

23 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:???

24 - 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 (~ nm) Limitations: Absence of is not definitive. Due to a long rotational correlation time of GFP (~18ns) no exact distance information can be obtained.

25 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), Xia & Liu, Biophys J (2001), Sorkin et al., Curr Biol (2000), Lorenz et al., Curr Biol (2004), 697.