Super-resolution Methods I - PALM
Detecting A Single Fluorescent Molecule? • Size: ~ 1nm • Absorption Cross-section: ~ 10-16 cm2 • Quantum Yield: ~1 Absorbance of 1 molecule = ? How many fluorescence photons per excitation photons?
De-convolution Microscopy Thompson, RE; Larson, DR; Webb, WW, Biophys. J. 2002,
Photo-activation De-convolution Accuracy # of photons
Photo-switchable Fluorescent Protein Gurskaya NG et al. 2006 Nat. Biotechnol.
What Next? • Z-resolution • Better fluorescent proteins • Multiple-color labeling • Cryo-temperature imaging
Super-Resolution: Beyond Diffraction Limit of λ/2: Near-Field: Distance <<Optical Wavelength Light not yet diffracted at sample Resolution not diffraction Limited, no diffraction, Limited by aperture size Aperture Diameter<<Wavelength: 50-100 nm Aperture-Surface distance<<Wavelength: 20 nm Probes made from pulled fiber-optics
Experimental Geometries with Fiber-based Probes trans epi • 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)
Fabrication of Tapered Fiber tips: cannot with standard pipette puller for electrophysiology CO2 Laser Pull-solenoid Pull down to 30-100 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 tips with Evaporated aluminum Coating confines light Rotate at magic angle For even coverage Bell Jar Hallen lab, NC State
Signal Strength vs Resolution Resolution only depends on aperture, not wavelength Theoretical: 1/r6 scaling 50 nm practical limit: 106 throughout loss of laser Hallen lab, NC State
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
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 Saykally, J. Phys. Chem. B, (2002)
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
Practical Concerns Hallen lab, NC State ↑ - Coating can have small pinholes: Loss of confinement - Easily damaged in experiment
Principle of the Apertureless NSOM Sharp tip of a electric conductor enhance (condense) the local electric field.
Stimulated Emission Rate: Absorption Rate: Number of atoms or molecules in lower energy level (Unit: per cm3) Number of atoms or molecules in lower energy level (Unit: per cm3) -σ12FN1 -σ21FN2 Absorption Cross-Section Units → cm2 Photon Flux Units → #/cm2sec Stimulated emission Cross-Section Units → cm2 (typical value ~ 10-19 to 10-18 cm2) Photon Flux Units → #/cm2sec σ12 = σ21
Stimulated Emission Depletion (STED) Drive down to ground state with second “dump”pulse, Before molecule can fluoresce Quench fluorescence and Combine with spatial control to make “donut”, achieve super-resolution in 3D (unlike NSOM)
STED Experimental Setup and PSF’s 100 nm Axial and lateral PSFs Need two tunable lasers, Overlapped spatially, temporally And synchronized Hell et al
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
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 2-photon 4pi With sidelobes gone