More Non-linear Microscopy

Nonlinear Polarization

P = aE + dE

2

+ d'E

3

+ . . .

E x = E xo cos

(2 p

ft

) =

E xo cos

( w

t

)

P x

=

aE xo cos

( w

t

) +

dE 2 xo cos 2

( w

t

) cos 2 (  )  1  cos( 2  ) 2

First experimental observation of SHG (1962) Right after invention of ruby laser

Coherent Anti-Stokes Raman Scattering (CARS)

w

as

 2 w

p

 w

s

850 nm Laser scanning Nonlinear optical microscope Collect SHG, TPEF Simultaneously Or TPEF

SHG is coherent: Forward propagates

Video Rate epi- CARS for Tissue Imaging Evans and Xie

TPEF and SHG Photophysics

Intensity ~ Power 2 Intensity ~ concentration Lifetime ~ ns Stoke shift Intensity ~ Power 2 Intensity ~ concentration 2 Instantaneous No loss of energy

TPEF and SHG “Spectra” TPEF 1) SHG “spectrum” mirrors Two-photon spectrum: Resonance enhancement 2) TPEF intensity >> SHG

Mertz, BJ 2001

Dipole Emission Patterns for scattering particles

Xie J. Phys. Chem l

Many dipoles aligned: All forward directed

Only forward direction for objects axially longer than wavelength

Calculations show that SHG emitted in two lobes

Mertz, BJ 2001

Forward/Backward Fractions SHG in Tendon: Wild type vs OI WT OI/OI Fw Fw Bw Bw WT larger fibers, also brighter, F/B Ratio?

Real Tissue: Only Scattering Matters Thick tissues, initial photon is multiply scatttered Some goes backwards direction Like SHG Backscattered collection A

image plane 60 50 40 30 20 10 0 0

B

50 150 200 250

Red, Black: experimental and Monte Carlo simulations From intralipid phantom Blue=mouse

CARS Image of Hairless Mouse Ear A Corneacytes

0 m m deep

D

100 m m 70 m m deep

E

100 m m 100 m m deep 100 m m

Adipocytes B Sebaceous glands

30 m m deep

C F

100 m m 100 m m 50 m m 20 m m 50 m m

Evans and Xie PNAS