1. I - PALM Super-resolution Methods

2. Detecting A Single Fluorescent Molecule? Size: ~ 1nm Absorption Cross-section: ~ 10 -16 cm 2 Quantum Yield: ~1 Absorbance of 1 molecule = ? How many fluorescence photons per excitation photons?

3. Single Molecule “Blinks”

5. Myosin V -- a motor protein.

7. De-convolution Microscopy Thompson, RE; Larson, DR; Webb, WW, Biophys. J. 2002,

8. Paul Selvin

9. Photo-activation De-convolution # of photons Accuracy

10. Photo-switchable Fluorescent Protein Gurskaya NG et al. 2006 Nat. Biotechnol.

11. Photo-activation Localization Microscopy (PALM)

12. stochastic optical reconstruction microscopy STORM

14. Ground-State Depletion (GSDIM)

16. What Next? Z-resolution Better fluorescent proteins Multiple-color labeling Cryo-temperature imaging

17. II. NSOM

18. Super-Resolution: Beyond Diffraction Limit of λ/2: Near-Field: Distance <<Optical Wavelength Aperture Diameter<<Wavelength: 50-100 nm Aperture-Surface distance<<Wavelength: 20 nm Probes made from pulled fiber-optics Resolution not diffraction Limited, no diffraction, Limited by aperture size Light not yet diffracted at sample

19. Transmission mode most common (far-field collection) Epi-illumination good for two-photon excitation Far-field excitation, Near field Collection mode good for SHG (not shown here) Experimental Geometries with Fiber-based Probes trans epi

20. Fabrication of Tapered Fiber tips: cannot with standard pipette puller for electrophysiology CO 2 Laser Pull-solenoid Pull down to 30-100 nm diameter Very fragile, fabrication not highly reproducible

21. EM of Uncoated Tip Hallen lab, NC State Uncoated tips do not confine light well for one photon excitation Good for NLO modes (intrinsic peak power confinement) Much higher transmission than coated tips

22. Coating confines light Hallen lab, NC State Coating tips with Evaporated aluminum Rotate at magic angle For even coverage Bell Jar

24. Signal Strength vs Resolution Theoretical: 1/r 6 scaling Hallen lab, NC State 50 nm practical limit: 10 6 throughout loss of laser Resolution only depends on aperture, not wavelength

25. Measuring forces Scanning Probe Feedback Mechanism: AFM and NSOM same implementation Need constant tip-specimen distance for near-field Use second NIR laser and 2-4 Sectored position sensitive diode Probe has mirror on top

26. Experimental Geometry with AFM type Feedback Tapered fibers use same Feedback as AFM Control piezo for Axial control

27. Nanonics Design Sits on Inverted Microscope Far-field collection

28. Saykally, J. Phys. Chem. B, (2002) Nonlinear excitation and NSOM with probe collection Use uncoated probes: Higher efficiency Metals can interact with Strong laser field, perturb sample (e.g. quench fluorescence) Confinement from NLO Don’t need coating Far-field excitation, NSOM collection

29. Shear force (topography), transmission NSOM, and fluorescence NSOM images of a phase separated polymer blend sample (NIST)

30. Limitations Shallow depth of view. Weak signal Very difficult to work on cells, or other soft samples Complex contrast mechanism – image interpretation not always straightforward Scanning speed unlikely to see much improvement

31. Hallen lab, NC State - Coating can have small pinholes: Loss of confinement - Easily damaged in experiment ↑ Practical Concerns

32. Aperture vs Apertureless NSOM

33. Sharp tip of a electric conductor enhance (condense) the local electric field. Principle of the Apertureless NSOM

34. Raman spectrum (SERRS) of Rh6G with and without AFM tip

35. Apertureless NSOM Probes

36. III. STED

38. Absorption Rate: -σ 12 FN 1 Absorption Cross-Section Units → cm2 Photon Flux Units → #/cm 2 sec Number of atoms or molecules in lower energy level (Unit: per cm 3 ) Stimulated Emission Rate: -σ 21 FN 2 Stimulated emission Cross-Section Units → cm2 (typical value ~ 10 -19 to 10 -18 cm 2 ) Photon Flux Units → #/cm 2 sec Number of atoms or molecules in lower energy level (Unit: per cm 3 ) σ12 = σ21

39. Stimulated Emission Depletion (STED) Quench fluorescence and Combine with spatial control to make “donut”, achieve super-resolution in 3D (unlike NSOM) Drive down to ground state with second “dump”pulse, Before molecule can fluoresce

41. Setup

42. STED Experimental Setup and PSF’s 100 nm Axial and lateral PSFs Need two tunable lasers, Overlapped spatially, temporally And synchronized Hell et al

43. Resolution increase with STED microscopy applied to synaptic vesicles

44. The real physical reason for the breaking of the diffraction barrier is not the fact that fluorescence is inhibited, but the saturation (of the fluorescence reduction). Fluorescence reduction alone would not help since the focused STED- pulse is also diffraction-limited.

45. RESOLFT: Extending the STED Idea Triplet – Singlet PAFP Photochromic Dye

46. 4-pi Microscopy

47. 4pi Microscopy: Improves Axial Resolution Excite high NA top and bottom

48. Standing Wave interference makes sidelobes Need deconvolution to remove sidelobes from image

49. The resolution is largely given by the extent of the effective 4Pi- spot, which is 3-5 times sharper than the spot of a regular confocal microscope

50. ~100 nm Axial Resolution 2-photon confocal 2-photon 4pi With sidelobes gone

51. GFP-labeled mitochondrial compartment of live Saccharomyces cerevisiae. 4-pi scope readily works for cell imaging

52. Combine STED with 4 pi for improved 3D resolution Over STED or 4Pi alone

55. 30 nm Resolution: 15 fold improvement over Diffraction Limit

56. Comparing to Confocal