I - PALM Super-resolution Methods. Detecting A Single Fluorescent Molecule? Size: ~ 1nm Absorption Cross-section: ~ 10 -16 cm 2 Quantum Yield: ~1 Absorbance.

I - PALM

Super-resolution Methods

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?

Single Molecule “Blinks”

Myosin V -- a motor protein.

De-convolution Microscopy

Thompson, RE; Larson, DR; Webb, WW, Biophys. J. 2002,

Paul Selvin

Nas /)12/( 22

Photo-activation De-convolution

# of photons

Accuracy

Photo-switchable Fluorescent Protein

Gurskaya NG et al. 2006 Nat. Biotechnol.

Photo-activation Localization Microscopy (PALM)

stochastic optical reconstruction microscopySTORM

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: 50-100 nmAperture-Surface distance<<Wavelength: 20 nmProbes made from pulled fiber-optics

Resolution not diffractionLimited, 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

transepi

Fabrication of Tapered Fiber tips: cannot with standard pipette puller for electrophysiology

CO2 Laser

Pull-solenoid

Pull down to 30-100 nm diameterVery fragile, fabrication not highly reproducible

EM of Uncoated Tip

Hallen lab, NC State

Uncoated tips do not confine light wellfor 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 withEvaporated aluminum

Rotate at magic angleFor even coverage

Bell Jar

Signal Strength vs Resolution

Theoretical: 1/r6 scaling

Hallen lab, NC State

50 nm practical limit: 106 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 2-4Sectored position sensitive diodeProbe has mirror on top

Experimental Geometry with AFM type Feedback

Tapered fibers use sameFeedback as AFMControl piezo for Axial control

Nanonics Design

Sits onInvertedMicroscopeFar-fieldcollection

Saykally, J. Phys. Chem. B, (2002)

Nonlinear excitation and NSOM with probe collection

Use uncoated probes:

•Higher efficiency

•Metals can interact withStrong laser field, perturb sample(e.g. quench fluorescence)

Confinement from NLODon’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:

-σ12FN1

AbsorptionCross-SectionUnits → cm2

Photon FluxUnits → #/cm2sec

Number of atoms ormolecules in lowerenergy level (Unit: per cm3)

Stimulated Emission Rate:

-σ21FN2

Stimulated emissionCross-SectionUnits → cm2(typical value ~ 10-19

to 10-18 cm2)

Photon FluxUnits → #/cm2sec

Number of atoms ormolecules in lowerenergy level (Unit: per cm3)

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 nmAxial and lateralPSFs

Need two tunable lasers, Overlapped spatially, temporallyAnd 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

2-photon 4piWith 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 resolutionOver STED or 4Pi alone

30 nm Resolution: 15 fold improvement over Diffraction Limit

Comparing to Confocal