1. Live tissue imaging (cornea) Optical imaging of tissue  cells Histology and Electron Microscopy can image ECM. Requires sectioning and staining. Stroma  Extracellular Matrix collagen is ubiquitous Light scatteringCellular fluorescence nonlinear microscopy  endogenous nonlinear optical signals High Resolution Imaging

2. Cornea (Rabbit) Epithelium Stroma Endothelium 100  m

3. 14  m

4. Corneal Stroma 30  m x y z y x z x y z 28 x 32 x 33  m

5. Intensity (a.u.) Wavelength (nm) 750 nm Intensity (a.u.) 800 nm 8  m 0 350400450500550 600 0 Corneal imaging signals

6. Spectral filtering of imaging signals 520 nm 400 nm 8  m keratocytes 30  m TPEF + SHG TPEFSHG

7. SHG in Collagen Collagen fiber quaternary structure tertiary structure ~

8. Polarization dependence of SHG E x (t) E y (t) P(t) x y z Collagen fiber Fundamental SHG

9. SHG Polarization Dependence Incident laser polarization 8  m

10. Optical characterization of tissue Polarization dependence of SHG in collagen Endogenous nonlinear optical signals to characterize tissue Linear (circular) polarization Spectroscopy of nonlinear optical signals to enhance contrast constituent specific imaging sensitive to long range order of collagen molecules

11. Cornea (diagnosis window to the eye) Wilmer Eye Institute, Johns Hopkins University U.S. 3 million cases of glaucoma U.S. 1.5 million diagnosed OHT and treated to reduce IOP Applanation Tonometry Corneal tissue properties affect IOP measurement central cornea thickness low tension glaucoma OHT 56% NLOM: characterize tissue material properties dependence on IOP