1. More Non-linear Microscopy

2. 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 3 +... Nonlinear Polarization

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

4. Coherent Anti-Stokes Raman Scattering (CARS)

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

6. Evans and Xie Video Rate epi- CARS for Tissue Imaging

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

9. Mertz, BJ 2001 1)SHG “spectrum” mirrors Two-photon spectrum: Resonance enhancement 2) TPEF intensity >> SHG TPEF TPEF and SHG “Spectra”

10. 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

11. Mertz, BJ 2001 Calculations show that SHG emitted in two lobes

12. 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?

13. image plane A 050100150200250 0 10 20 30 40 50 60 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

14. 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