Abstract
A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.
Highlights
Laser Microbeam and Medical Program, Beckman Laser Institute and Center for Biomedical Engineering, University of California, Irvine, California 92612
The multiphoton microscopy (MPM) system used in this Letter employs a Ti:Al2O3 oscillator pumped by a frequency-doubled Nd:YVO4 solid-state laser.[8]
The cornea is composed of well-defined layers, including a surface of epithelial cells, the stroma, and an underlying endothelial layer
Summary
Received June 12, 2002 A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited f luorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Biomedical imaging using multiphoton microscopy (MPM) is a rapidly growing field.[1,2] Most MPM studies employ two-photon excited f luorescence (TPF) to form images.[3] More recently, harmonic generation[4,5] and stimulated Raman[6,7] signals have been used to image cells and tissues.
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