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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?

Single Molecule Blinks

Myosin V -- a motor protein.

De-convolution Microscopy

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

Paul Selvin

Accuracy

( s 2 a 2 / 12) / N
# of photons

Photo-activation

De-convolution

Photo-switchable Fluorescent Protein

Gurskaya NG et al. 2006 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 /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

Scanning Probe Feedback Mechanism:


AFM and NSOM same implementation
Need constant tip-specimen distance for near-field

Measuring
forces

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
Dont 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

Aperture vs Apertureless NSOM

Principle of the Apertureless NSOM

Sharp tip of a electric conductor enhance (condense)


the local electric field.

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

Apertureless NSOM Probes

III. STED

Stimulated Emission Rate:

Absorption Rate:

-12FN1
Absorption
Cross-Section
Units cm2

Number of atoms or
molecules in lower
energy level (Unit:
per cm3)

Photon Flux
Units #/cm2sec

-21FN2
Stimulated emission
Cross-Section
Units cm2
(typical value ~ 10-19
to 10-18 cm2)

12 = 21

Number of atoms or
molecules in lower
energy level (Unit:
per cm3)

Photon Flux
Units #/cm2sec

Stimulated Emission Depletion (STED)

Drive down to ground state with second dumppulse,


Before molecule can fluoresce
Quench fluorescence and Combine with spatial control
to make donut, achieve super-resolution in 3D (unlike NSOM)

Setup

STED Experimental Setup and PSFs

100 nm
Axial and lateral
PSFs

Need two tunable lasers,


Overlapped spatially, temporally
Hell et al
And synchronized

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 STEDpulse 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 4Pispot, 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

4-pi scope readily works for cell imaging

GFP-labeled mitochondrial compartment of live


Saccharomyces cerevisiae.

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

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