Post on 25-Dec-2015
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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?
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
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
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
Sharp tip of a electric conductor enhance (condense) the local electric field.
Principle of the Apertureless NSOM
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
STED Experimental Setup and PSF’s
100 nmAxial and lateralPSFs
Need two tunable lasers, Overlapped spatially, temporallyAnd 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.
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