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

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 1) 서구 사회의 학교 제도 - schola : 한가, 여가를 뜻함, 오늘날의 학교 school - 고대 그리스 사회에서 지배계급의 지위와 신분을 유 지하기 위해 소수의 귀족계급을 위해 조직되어 교육 실시 - 중세 유럽사회의 학교는 소수의 성직자나 지도자 양 성 을 위한 교회부속의 사원 학교가 대부분

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

8. FRET  Distance dependent interaction between the electronic excited states of two molecules * not sensitive to the surrounding solvent shell of a fluorophore *Donor-Acceptor 의 Energy transfer 는 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 DonorAcceptorRo(Ǻ) FluoresceinTetramethlrhoda mine 55 IAEDANSFluorescein46 EDANSDabcyl33 FluoresceinFluoresscein44 BODIPY FL 57 FluoresceinQsy7&Qsy9 dyes 61

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

13. FRET  Förster Equation 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. Inter-molecular FRET Intra-molecular FRET Biological application using FRET (ex: cameleon)

18. FRET  Biological application using FRET

19. Outline 1.What is fluorescence?? 2.Fluorescent molecules 3.Equipment for single-molecule fluorescence experiments 4.Some applications & examples

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

21. S0S0 S1S1 T0T0 transition involving emission/absorption of photon radiationless transition absorption +hν fluorescence -hν internal conversion intersystem crossing internal conversion 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 k nr krkr S0S0 S1S1

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 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) 550 580

27. extinction coefficient (  ):~100 000 M -1 cm -1 the fluorescence of a single TMR can be measured easily absorption cross section (  )  =  · 2303 / N 0 :~4·10 -16 cm 2 excitation power: ~100 W/cm 2 excitation photon flux = power / photon energy:~2.5 · 10 20 photons·s -1 ·cm -2 photon energy = h·c/ #excitations·molecule -1 ·s -1 #exc = flux·  ~10 5 photons·s -1 ·cm -2  = area of an opaque object with the same that blocks the light as good as the molecule dI/I = (  ·C·N Av /1000)·dL dI/I =  ·2.303·dL #emitted photons·molecule -1 ·s -1 #em = #exc·QY ~10 5 photons·s -1 ·cm -2

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

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

33. Solvent effects Non-polar solvent Polar solvent Energy Static molecular dipole moment S0S0 S1S1 S1S1 S0S0 S1S1

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

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

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

37. Transfer Efficiency Fraction of excitations transferred to acceptor R 0 = Förster radius, maximum 10 nm for large overlap

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

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. multicolor 2-photon microscopy

42. specific labeling with various colors

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

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