An optically sectioning ocular fluorometer microscope is described with the capability of measuring the emission spectra of molecules in planes along the microscope axis. Its unique feature is that the objective is attached to a piezoelectric driver and scans from the tear film to the aqueous humor. This permits measurements on living animals and adoption for clinical use. The excitation light from a laser (nitrogen, dye, argon or helium cadmium) couples to the microscope via a quartz optical fiber. The light is projected through a 100μm slit on the excitation side, through one half of the objective. The emitted light is collected by the second half of the objective and passes a second 100μm slit in the conjugate plane of the eyepiece. The depth resolution is 6μm with an 100× objective, and 18μm with a 50 power objective. The fluorescence is coupled by a quartz fiber to an optical spectrum analyzer. It consists of a monochromator with two microchannel plates attached to a linear diode array. The photocathode of the detector is gated for use with pulsed lasers or it can operate in the continuous mode. The applications include fluorescence measurements on thin layered structures. The present study involves the noninvasive measurement of oxidative metabolism of the component layers of the in vivo cornea. This is based on fluorescence measurements of the reduced pyridine nucleotide in the cornea. The fluorescence signals from the corneal epithelial (30μm) and endothelial (4μm) are clearly defined. Other applications to ophthalmology include studies of the fluorescence form the component layers of the ocular lens. Support from N.I.I. EY06958.
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