Fluorescence Resonance Energy Transfer (FRET)

1. Fluorescence Resonance Energy Transfer (FRET)

2. FRET • Resonance energy transfer can occur when the donor and acceptor molecules are less than 100 A of one another • Energy transfer is non-radiative which means the donor is not emitting a photon which is absorbed by the acceptor • Fluorescence RET (FRET) can be used to spectrally shift the fluorescence emission of a molecular combination. Resonance Energy Transfer

3. FRET • The mechanism of FRET involves a donor fluorophore in an excited electronic state, which may transfer its excitation energy to a nearby acceptor chromophore • non-radiative fashion through long-range dipole-dipole interactions

4. J(λ) Fluorescnece Intensity Donor fluorescnece Acceptor absorption Wavelength FRET • The absorption spectrum of the acceptor must overlap fluorescence emission spectrum of the donor

5. FRET • Energy Donor excitation state Emission Acceptor excitation state

6. 학교 제도 : 교육 제도 중 학교에 관한 제도 사회적으로 가장 먼저 공인된 제도, 형식적 교육 제도 • 서구 사회의 학교 제도 • - schola : 한가, 여가를 뜻함, 오늘날의 학교 school • -고대 그리스 사회에서 지배계급의 지위와 신분을 유 • 지하기위해 소수의 귀족계급을 위해 조직되어 교육 • 실시 • -중세 유럽사회의 학교는 소수의 성직자나 지도자양 • 성을 위한 교회부속의 사원 학교가 대부분

7. 488nm light excitation excitation FITC 520nm light TRITC TRITC FITC 630nm light FRET

8. FRET • Distance dependent interaction between the electronic excited states of two molecules *not sensitive to the surrounding solvent shell of a fluorophore *Donor-Acceptor의 Energy transfer는 거리에 의해 효율이 결정 (~10nm) • Spectral properties of involved chromophore

9. FRET • Calculation Efficiency of Energy Transfer = E = kT/(kT + kf + k’) kT = rate of transfer of excitation energy kf = rate of fluorescence k’ = sum of the rates of all other deexcitation processes E = R60/ R60+ R6

10. FRET • Förster Equation Ro= Forster radius = Distance at which energy transfer is 50% efficient = 9.78 x 103(n-4*fd*k2*J)1/6 Å fd-fluorescence quantum yield of the donor in the absence of acceptor n- the refractive index of the solution k2- the dipole angular orientation of each molecule j- the spectral overlap integral of the donor and acceptor

11. Typical values of R0

12. FRET Critical Distance for Common RET Donor-Acceptor Pairs

13. FörsterEquation FRET • Förster Equation

14. FRET Schematic diagram of FRET phenomena

15. FRET SUMMARY • Emission of the donor must overlap absorbance of the acceptor • Detect proximity of two fluorophores upon binding • Energy transfer detected at 10-80Ǻ

16. FRET

17. FRET Biological application using FRET (ex: cameleon) Intra-molecular FRET Inter-molecular FRET

18. FRET • Biological application using FRET

19. Outline • What is fluorescence?? • Fluorescent molecules • Equipment for single-molecule fluorescence experiments • Some applications & examples

20. fluorescence from molecules physical fundaments photon molecule in excited state molecule in ground state photon light can induce transitions between electronic states in a molecule

21. radiationless transition absorption internal conversion fluorescence intersystem crossing S1 T0 -hν +hν internal conversion S0 transition involving emission/absorption of photon fluorescence the Jablonski diagram

22. fluorescence properties that can be measured • spectra (environmental effects) • fluorescence life times • polarization (orientation and dynamics) • excitation transfer (distances -> dynamics) • location of fluorescence

23. fluorescence requirements for a good fluorophore • good spectral properties • strong absorber of light (large extinction coefficient) • high fluorescence quantum yield • low quantum yield for loss processes (triplets) • low quantum yield of photodestruction • small molecule / easily attachable to biomolecule to be studied

24. 1.7 Fluorescence quantum yield S1 knr kr S0

25. fluorescence chromophores: intrinsic or synthetic?? • common intrinsic fluorophores like tryptophan, NAD(P)H are not good enough • chlorophylls & flavins work • in most cases extrinsic fluorophores have to be added: • genetically encoded (green fluorescence protein) • chemical attachment of synthetic dyes

26. fluorescence a typical synthetic chromophore: tetramethylrhodamine 580 550 • extinction coefficient: ~100,000 Molar-1 cm-1 • fluorescence quantum yield: ~50% • triplet quantum yield <1% • available in reactive forms (to attach to amines, thiols) and attached to many proteins and other compounds (lipids, ligands to proteins)

27. the fluorescence of a single TMR can be measured easily extinction coefficient (e): ~100 000 M-1 cm-1 • = area of an opaque object with the same that blocks the light as good as the molecule dI/I = (s·C·NAv/1000)·dL dI/I = e·2.303·dL absorption cross section (s) s = e · 2303 / N0: ~4·10-16 cm2 excitation power: ~100 W/cm2 excitation photon flux = power / photon energy: ~2.5 · 1020 photons·s -1·cm-2 photon energy = h·c/l #excitations·molecule-1·s-1 #exc = flux·s ~105 photons·s -1·cm-2 #emitted photons·molecule-1·s-1 #em = #exc·QY ~105 photons·s -1·cm-2

28. single-molecule fluorescence microscopy • excitation source: laser • Lasers cw (ion), pulsed (Nd-YAG, Ti-sapphire, diodes • optical system with high collection efficiency: high NA objective • optics to separate fluorescence from excitation light: filters / dichroic mirrors • monochromators, spectrographs; filters: colored glass, notch holographic, multidielectric • detector: - CCD camera, PMT - eyes; PMT, APD, CCDPhotoMultiplier Tube, Avalanche PhotoDiode, • Charge Coupling Device (signal is usually weak) + electronics

29. rotation of F1-ATPase • Adachi, K., R. Yasuda, H. Noji, H. Itoh, Y. Harada, M. Yoshida, and K. Kinosita, Jr. 2000. Proc. Natl. Acad. Sci. U.S.A. 97:7243-7247

30. folding / unfolding of RNA (Tetrahymena ribozymes) X. Zhuang, L. Bartley, H. Babcock, R. Russell, T. Ha, D. Herschlag, and S. Chu Science 2000 June 16; 288: 2048-2051.

31. FLUORESCENCE MEASUREMENTS • Information given by each property of fluorescence photons:- spectrum- delay after excitation (lifetime)- polarization

32. Spectra Fluo. intensity Sample Detector Laser exc fluo exc fluo Spectrograph Excitation spectrum Fluorescence spectrum

33. Solvent effects Energy Non-polar solvent Polar solvent S1 S1 S1 S0 S0 Static molecular dipole moment

34. Fluorescence Lifetime Sample number Pulsed laser Filter delay, t Detector Laser pulses time delay photons

35. Polarization Fluid Rigid depolarized polarized Polarization memory during the fluorescence lifetime : fluo. anisotropy

36. Fluorescence Resonance Energy Transfer (FRET) Dipole-dipole interaction (near-field) Donor Acceptor

37. Transfer Efficiency • Fraction of excitations transferred to acceptor • R0 = Förster radius, maximum 10 nm for large overlap

38. R>10 nm R<10 nm Förster Resonance Energy Transfer

39. FRET studies of interaction and dynamics(molecular ruler) Association of two biomolecules Dynamics of a biomolecule

40. Other specific labeling and imaging • Possibility to specifically label certain biomolecules, sequences, etc. with fluorophores • Staining and imaging with various colors • Detection of minute amounts (DNA assays) • Fluorescence lifetime imaging (FLIM) • Fluorescence recovery after photobleaching

41. multicolor2-photonmicroscopy

42. specific labeling with various colors

43. I(t+ t) I(t) t t g(2) logt Fluorescence Correlation Spectroscopy Keeps track of the fluctuations of the fluorescence intensity.

44. Single molecule spectroscopy • Single molecule tracking • dynamics of single enzyme • sp-FRET • orientation fluctuations • lifetime measurement