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