I - PALM Super-resolution Methods

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

Single Molecule “Blinks”

Myosin V -- a motor protein.

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

Paul Selvin

Photo-activation De-convolution # of photons Accuracy

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

Photo-activation Localization Microscopy (PALM)

stochastic optical reconstruction microscopy STORM

Ground-State Depletion (GSDIM)

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

II. NSOM

Super-Resolution: Beyond Diffraction Limit of λ/2: Near-Field: Distance <<Optical Wavelength Aperture Diameter<<Wavelength: 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

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

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

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

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

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

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

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

Nanonics Design Sits on Inverted Microscope Far-field collection

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

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

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

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

Aperture vs Apertureless NSOM

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

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

Apertureless NSOM Probes

III. STED

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 ~ to cm 2 ) Photon Flux Units → #/cm 2 sec Number of atoms or molecules in lower energy level (Unit: per cm 3 ) σ12 = σ21

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

Setup

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

Resolution increase with STED microscopy applied to synaptic vesicles

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.

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

4-pi Microscopy

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

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

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

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

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

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

30 nm Resolution: 15 fold improvement over Diffraction Limit

Comparing to Confocal