Scanning Microscope For Optically Sectioning The Living Cornea

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Abstract
A prototype of a clinical optically sectioning, two-dimensional redox fluorescence imaging microscope is described. Ultraviolet light from an arc lamp is conducted to the optical system with a quartz optical fiber. A variable slit projects the light onto the cornea after passing an excitation interference filter. The dipping cone of the microscope applanates the cornea and focuses the light onto the endothelial cell layer which is 6 microns thick. The fluorescence emission from the mitochondrial reduced pyridine nucleotides (NADH + NADPH) is detected by a microchannel plate-gated intensifier attached to a Newvicon Video Camera and a digital image processor. The intrinsic natural cellular fluorescence is imaged and is indicative of the cellular state of oxidative metabolism. In addition, an optically sectioning microscope was developed with an optical spectrum analyzer to characterize the fluorescence spectra from thin layers in the cornea and ocular lens. Its unique feature is that the scanning objective is attached to a piezoelectric driver and scans the eye from the tear film to the aqueous humor. The depth resolution is 6 microns with an 100 x objective and 18 microns with a 50 power objective (100 micron slits). The applications include fluorescence measurements on biological layered structures. The present study involves the noninvasive measurement of oxidative metabolism of the component layers of the in vivo cornea. Finally, the utility of confocal microscopy in ophthalmology is demonstrated as a series of confocal images of the rabbit cornea with a depth resolution less than one micron.

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