More Non-linear Microscopy

1. More Non-linear Microscopy

2. Nonlinear Polarization P = aE + dE2 + d'E3 + . . . Ex = Exo cos(2pft) = Exo cos (wt) Px = aExo cos (w t) + dE2xo cos2 (w t)

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

4. Coherent Anti-Stokes Raman Scattering (CARS)

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

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

7. TPEF and SHG Photophysics Intensity ~ Power2 Intensity ~ concentration Lifetime ~ ns Stoke shift Intensity ~ Power2 Intensity ~ concentration2 Instantaneous No loss of energy

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

10. Dipole Emission Patterns for scattering particles Xie J. Phys. Chem Many dipoles aligned: All forward directed l Only forward direction for objects axially longer than wavelength

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

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

13. 60 A B 50 Percent Scattered Back 40 30 image plane 20 10 0 0 50 100 150 200 250 Thickness (µm) Real Tissue: Only Scattering Matters 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

14. 0 mm deep 30 mm deep A B C 50 mm 70 mm deep D F 100 mm 100 mm deep E 50 mm 100 mm 100 mm 100 mm 100 mm 20 mm CARS Image of Hairless Mouse Ear Corneacytes Sebaceous glands Adipocytes Evans and Xie PNAS