ABSTRACT
This chapter presents a second harmonic generation (SHG)-based imaging paradigm called nonlinear optical high-resolution macroscopy, which allows for the generation of large-scale, high-resolution, three dimensional reconstructions of corneal cross sections. To generate SHG signals, ultrashort laser pulses, usually no longer than several hundred femtoseconds and with wavelengths in the near-infrared regime, are focused into a specimen through microscope optics. Using SHG imaging, individual lamellae can be imaged at submicron resolution. A breakthrough in optical imaging occurred with the first demonstration of nonlinear optical (NLO) imaging by W. Denk et al. using a subpicosecond pulsed red laser to molecularly excite ultraviolet photoactivated fluorophores by two photon excitation. A unique feature of NLO imaging is that multiphoton interactions are limited to the focal volume of the objective leading to increased axial resolution. Isolation and characterization of stromal lamellar structure using NLO- high-resolution macroscopy dramatically demonstrate the complexity of collagen organization in cornea that is not clearly depicted by conventional biomechanical models.
