Escolar Documentos
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I - PALM
Absorbance of 1 molecule = ?
How many fluorescence photons per excitation photons?
De-convolution Microscopy
Paul Selvin
Accuracy
( s 2 a 2 / 12) / N
# of photons
Photo-activation
De-convolution
Photo-activation Localization
Microscopy (PALM)
What Next?
Z-resolution
Better fluorescent proteins
Multiple-color labeling
Cryo-temperature imaging
II. NSOM
trans
epi
Pull-solenoid
EM of Uncoated Tip
Bell Jar
Hallen lab, NC State
Measuring
forces
Nanonics Design
Sits on
Inverted
Microscope
Far-field
collection
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
III. STED
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
Setup
100 nm
Axial and lateral
PSFs
Triplet Singlet
PAFP
Photochromic Dye
4-pi Microscopy
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
2-photon confocal
2-photon 4pi
2-photon 4pi
With sidelobes
gone
Comparing to Confocal