Ultraviolet confocal fluorescence microscopy of the in vitro cornea: redox metabolic imaging

Abstract

A laser scanning microscope was fitted with two argon-ion lasers that provided wavelengths in the regions of 364, 488, and 514 nm. A Zeiss water objective of 25 x , with a numerical aperture of 0.8, corrected for the UV, was used to measure the fluorescence from optical sections of freshly enucleated rabbit eyes. The confocal microscope was used in both the reflected and fluorescent modes to image in situ epithelial and endothelial cells. An excitation wavelength of 364 nm and emission at 400-500 nm were used to image the fluorescence from reduced pyridine nucleotides. We demonstrate the feasibility of two-dimensional fluorescent confocal imaging of reduced pyridine nucleotides in corneal epithelial and endothelial cells.