More Non-linear Microscopy

E x = E xo cos(2  ft) = E xo cos (   t) P x = aE xo cos (  t) + dE 2 xo cos 2 (  t) P = aE + dE 2 + d'E Nonlinear Polarization

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

Coherent Anti-Stokes Raman Scattering (CARS)

Collect SHG, TPEF Simultaneously Laser scanning Nonlinear optical microscope 850 nm SHG is coherent: Forward propagates Or TPEF

Evans and Xie Video Rate epi- CARS for Tissue Imaging

Intensity ~ Power 2 Intensity ~ concentration Lifetime ~ ns Stoke shift Intensity ~ Power 2 Intensity ~ concentration 2 Instantaneous No loss of energy TPEF and SHG Photophysics

Mertz, BJ )SHG “spectrum” mirrors Two-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 that SHG 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 Percent Scattered Back Thickness (µm) B Thick tissues, initial photon is multiply scatttered Some goes backwards direction Like SHG Backscattered collection Red, Black: experimental and Monte Carlo simulations From intralipid phantom Blue=mouse Real Tissue: Only Scattering Matters

100  m 50  m 100  m 20  m 30  m deep 70  m deep 100  m deep 0  m deep ABC D E F 50  m CorneacytesSebaceous glands Adipocytes Evans and Xie PNAS CARS Image of Hairless Mouse Ear