Bi/BE 177: Principles of Modern Microscopy

1. Bi/BE 177: Principles of Modern Microscopy Lecture 15: FRET, TIRFM, Super-resolution microscopy Part I Andres Collazo, Director Biological Imaging Facility Ke Ding, Graduate Student, TA Wan-Rong (Sandy) Wong, Graduate Student, TA

2. Lecture 15: FRET, TIRF, NSOM • FRET • FLIM review • Total internal reflection fluorescence microscopy (TIRFM) • Super resolution microscopy • NSOM • Critiquing figures

3. Questions about last lecture?

4. Förster Resonance Energy Transfer (FRET) • Great method for the detection of: • Protein-protein interactions • Enzymatic activity • Small molecules interacting inside a cell

5. Remember our fluorescence discussion? Resonance Energy Transfer (non-radiative) The Bad: Self-quenching If dye at high concentration “hot-potato” the energy until lost

6. ~0.1uM Log I Log [dye] • Resonance Energy Transfer (non-radiative) “Self-quenching” of dye (“hot-potato” the energy until lost) Depends on: Dye Concentration Geometry Environment

7. FRET: Resonance Energy Transfer (non-radiative) The Good: FRET as a molecular yardstick Transfer of energy from one dye to another Depends on: Spectral overlap Distance Alignment

8. donor acceptor FRET: Optimize spectral overlap Optimize k2-- alignment of dipoles Minimize direct excitement of the acceptor (extra challenge for filter design)

9. Non-radiative transfer -xx- Less -xx- Less FRET Diagram 4nsec 0.8 emitted

10. KT = (1/τD) • [R0/r]6 The Förster Equations. R0 = 2.11 × 10-2 • [κ2 • J(λ) • η-4 • QD]1/6 eA J(λ) r is the center-to-center distance (in cm) between the donor and acceptor tD is the fluorescence lifetime of the donor in the absence of FRET k2 is the dipole-dipole orientation factor, QD is the quantum yield of the donor in the absence of the acceptor  is the refractive index of the intervening medium, FD (l) is the fluorescence emission intensity at a given wavelength l (in cm) eA (l) is the extinction coefficient of the acceptor (in cm -1 M -1). The orientation factor k2 can vary between 0 and 4, but typically k2 = 2/3 for randomly oriented molecules (Stryer, 1978). When r = R0, the efficiency of FRET is 50% (fluorescein-tetramethylrhodamine pair is 55 Å)

11. FRET: Resonance Energy Transfer (non-radiative) The Good: FRET as a molecular yardstick Transfer of energy from one dye to another Depends on: Spectral overlap Distance Alignment

12. Remember: Going back to our Fluorescence lecture How dipole affects FRET as a molecular yardstick Fluorescent Dye Dipole antenna Delocalized electrons Longer dipole, longer λ

13. Fluorescent dye as dipole antenna  • Absorption depends on orientation  E Propagation direction  E Propagation direction

14. Fluorescent dye as dipole antenna Noemission alongthedipoleaxis μE • Orientation of fluorescence emission Someemission alongthisdirection  Maximal emission normal tothe dipoleaxis Maximal emission normal tothe dipoleaxis Noemission alongthedipoleaxis Dipole radiation pattern

15. Fluorescent dye as dipole antenna • Orientation of fluorescence emission affects FRET efficiency

16. Isolated donor Donor distance too great Donor distance correct More about FRET (Förster Resonance Energy Transfer) Effective between 10-100 Å only Emission and excitation spectrum must significantly overlap Note: donor transfers non-radiatively to the acceptor From J. Paul Robinson, Purdue University

17. FRET efficiency and the Förster Equations • Distance between donor and acceptor • When r = R0, the efficiency of FRET is 50% • When R <R0, EFRET > 0.50 • When R > R0, EFRET < 0.50 KT = (1/τD) • [R0/r]6 R0 = 2.11 × 10-2 • [κ2 • J(λ) • η-4 • QD]1/6 eA J(λ)

18. Optimizing FRET: Designs of new FRET pairs • Difficult to find two FRET pairs that can use in same cell • Used as Caspase 3 biosensors and for ratiometric imaging

19. Fluorophore brightness =  Q DsRedQ ~ 0.79 x 75,000 ~ 59,250 M-1.cm-1 (100%) mRFP1 Q ~ 0.25 x 50,000 ~ 12,500 M-1.cm-1 (21%) eGFPQ ~ 0.6 x 55,000 ~ 33,000 M-1.cm-1 (56%) Fluorescein Q ~ 0.8 x 70,000 ~ 56,000 M-1.cm-1 (95%) (dye!)

20. Optimizing FRET: Designs of new FRET pairs • mAmetrine developed by directed protein evolution from violet excitable GFP variant • Bright, extinction coefficient = 44,800 M-1 cm-1 • Quantum yield = 0.58 • But bleaches, 42% of mCitrine time and 1.7% of tdTomato

21. 4nsec Problems with FRET • The acceptor excited directly by the exciting light • “FRET” signal with no exchange • Increased background • Decreases effective range for FRET assay

22. Problems with FRET 2. Hard to really serve as a molecular yardstick* • Orientation seldom known • assume k2= 2/3 (random assortment) • Exchange depends on environment of dipoles • Amount of FRET varies with the lifetime of the donor fluorophore * r = R0, the efficiency of FRET is 50% (fluorescein-tetramethylrhodamine pair is 55 Å)

23. 4nsec Amount of FRET varies with the lifetime of the donor fluorophore Longer lifetime of the donor gives longer time to permit the energy transfer (more for longer) Added Bonus: Allows lifetime detection to reject direct excitement of the acceptor (FRET=late)

24. Fluorescence Lifetime Imaging Microscopy (FLIM) • Measure spatial distribution of differences in the timing of fluorescence excitation of fluorophores • Combines microscopy with fluorescence spectroscopy • Fluorescent lifetimes very short (ns) so need fast excitation and/or fast detectors • Requirements for FLIM instruments • Excitation light intensity modulated or pulsed • Emitted fluorescence measured time resolved

25. Fluorescence Lifetime Imaging Microscopy (FLIM) • Two methods for FLIM • Frequency-domain • Intensity of excitation light continuously modulated • For emission measure phase shift & decrease in modulation • Time-domain • Pulsed excitation that is faster than fluorescence lifetime • Emission measurement is time-resolved

26. FRET and FLIM • Donor fluorescence lifetime during FRET reduced compared to control donor fluorescence lifetime • During FRET, donor fluorescence lifetime less than control donor fluorescence lifetime (tD) • But isn’t it easier to image decreases in donor fluorescence intensity rather than measure fluorescence lifetime? KT = (1/τD) • [R0/r]6

27. FRET and FLIM: addressing nonlinearities • Brightness (or intensity) of fluorophore, as measured on your image, more than just Q • Local concentration of fluorophore • Optical path of microscope • Local excitation light intensity • Local fluorescence detection efficiency • FLIM provides independent measure of local donor lifetime

28. Isolated donor Donor distance too great Donor distance correct FRET and FLIM measure different parameters • FRET • Donor versus Acceptor fluorescent intensity • FLIM • Lifetime of donor with or without acceptor present.

29. Going back to those problems with FRET:These drawbacks can all be used to make sensors Change in FRET for changes in: • Orientation • cameleon dye for Ca++ • Local environment • Phosphate near fluorophore • Membrane voltage (flash) • Change in lifetime of donor • Binding of molecule displacing water

30. Cameleon: FRET-based and genetically-encoded calcium probe Calmodulin bonds Ca2+ and changes its conformation [Ca2+] Miyawaki et al, Nature, 1997 Cameleon family: calmodulin-based indicators of [Ca2+] using FRET isosbestic point

31. Paper to read • Pearson, H., 2007. The good, the bad and the ugly. Nature 447, 138-140. • http://www.nature.com/nature/journal/v447/n7141/full/447138a.html

32. Single molecule tracking • High speed • Single molecule imaging • Fluorescence correlation spectroscopy (FCS) • Total internal reflection microscopy (TIRF) • Super-resolution qi qi Interface

33. Total internal reflection fluorescence (TIRF) microscopy • Technique that dominates most single molecule imaging approaches

34. Internal reflection depends on refractive index differences sin q critical = h1 / h2

35. Evanescent waves • Near-field phenomenon • Higher frequency, more information • Formed at boundary between two media with different wave motion properties • Evanescent waves quantum tunneling phenomenon • Product of Schrödinger wave equations Exponential decay

36. Metamaterials with negative refractive indices could be used to make superlenses for super resolution microcopy • Maxwell's fish-eye lens could do it with positive refractive indices • Refractive index changes across lens (blue shading) • Harness information on resolution from evanescent waves • Type of Luneburg lens • Tyc T, Zhang X (2011) Forum Optics: Perfect lenses in focus. Nature 480: 42-43.

37. TIRFM illumination configurations Prism method Objective Lens method Ideally NA of 1.45 or higher

38. TIRFM illumination configurations Prism method Objective Lens method This is the way to go But … • Restricts access to specimen (difficult to manipulate) • Most illuminate opposite objective so have to pass through specimen • If prism on same side then more complicated alignment

39. TIRFM applications • Benefits for imaging minute structures or single molecules in specimens with tons of fluorescence outside of optical plane of interest • Examples: Brownian motion of molecules in solution, vesicles undergoing endocytosis or exocytosis, or single protein trafficking in cells • Can get dramatic increase in signal-to-noise ratio from thin excitation region • Microsphere example

40. TIRFM applications • Ideal tool for investigation of both the mechanisms and dynamics of many of the proteins involved in cell-cell interactions • Live cell imaging • GFP-vinculin to see focal adhesions on coverslip

41. TIRFM applications • Single molecule imaging • Time lapse of GFP-Rac moving along filopodia • In fact, most single molecule imaging today done with TIRFM

42. TIRFM versus Confocal Microscopy • Confocal not limited to plane at interface, can go deeper • TIRFM has thinner optical section (100 nm vs 600 nm) • TIRFM, like two photon, only excites sample at focal plane • TIRFM is cheaper to implement than confocal • TIRFM is NOT super-resolution (except in Z)

43. Highly inclined and laminated optical sheet (HILO) microscopy • How to make TIRF microscope go deeper • Use a highly inclined thin illumination • Like TIRF a wide field technique

44. Spatial Resolution of Biological Imaging Techniques

45. Super-resolution microscopy • “True” super-resolution techniques • Subwavelength imaging • Capture information in evanescent waves • Quantum mechanical phenomenon • “Functional” super-resolution techniques • Deterministic • Exploit nonlinear responses of fluorophores • Stochastic • Exploit the complex temporal behaviors of fluorophores

46. Spatial Resolution of Biological Imaging Techniques “True” super-resolution “Functional”

47. Remember the different types of microscopy from previous lecture? • Wide-field microscopy • Illuminating whole field of view • Confocal microscopy • Spot scanning • Near-field microscopy • For super-resolution

48. Near-Field Scanning Optical Microscopy (NSOM) • Scanning Near-Field Optical Microscopy (SNOM) • Likely the super-resolution technique with the highest resolution • But only for superficial structures • A form of Scanning Probe Microscopy (SPM)

49. All the types of microscopes

50. Near-Field Scanning Optical Microscopy (NSOM)Break the diffraction limit by working in the near-field Launch light through small aperture Illuminated “spot” is smaller than diffraction limit (about the size of the tip for a distance equivalent to tip diameter) Near-field = distance of a couple of tip diameters