More Non-linear Microscopy
Ex = Exo cos(2 ft) = Exo cos (t)
Px = aExo cos (t) + dE2xo cos2 (t)
P = aE + dE2 + d'E3 + . . .
2
)2cos(1)(cos2
Nonlinear Polarization
First experimental observation of SHG (1962)
Right after invention of ruby laser
spas 2
Coherent Anti-Stokes Raman Scattering (CARS)
Collect SHG, TPEF Simultaneously
Laser scanning Nonlinear optical microscope850 nm
SHG is coherent:Forward propagates
Or TPEF
Evans and Xie
Video Rate epi- CARS for Tissue Imaging
Intensity ~ Power2
Intensity ~ concentrationLifetime ~ nsStoke shift
Intensity ~ Power2
Intensity ~ concentration2
InstantaneousNo loss of energy
TPEF and SHG Photophysics
Mertz, BJ 2001
1) SHG “spectrum” mirrorsTwo-photon spectrum:Resonance enhancement
2) TPEF intensity >> SHG
TPEF
TPEF and SHG “Spectra”
Only forward direction for objects axially longer than wavelength
Dipole Emission Patterns for scattering particles
Xie J. Phys. Chem
Many dipoles aligned:All forward directed
Mertz, BJ 2001
Calculations show thatSHG emitted in two lobes
WT OI/OI
Fw
Bw
Fw
Bw
Forward/Backward Fractions SHG in Tendon: Wild type vs OI
WT larger fibers, also brighter, F/B Ratio?
image plane
A
0 50 100 150 200 2500102030405060
Pe
rce
nt
Sca
tte
red
Ba
ckThickness (µm)
B
Thick tissues, initial photon is multiply scattteredSome goes backwards directionLike SHG Backscattered collection
Red, Black: experimental and Monte Carlo simulationsFrom intralipid phantomBlue=mouse
Real Tissue: Only Scattering Matters
100 m
100 m
50 m
100 m
100 m 100 m 20 m
30 m deep
70 m deep
100 m deep
0 m deepA B C
D
E
F50 m
Corneacytes Sebaceous glands
Adipocytes
Evans and XiePNAS
CARS Image of Hairless Mouse Ear
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