A microfluorometer for measuring diffusion of fluorophores across the cornea

A microscope has been modified to make a confocal spatially scanning fluorometer so that the concentration profile of a fluorophore across an isolated cornea can be measured. A slit of light is focused through one half of the objective to excite fluorescence. A confocal slit is placed at the image-plane of the microscope to collect the fluorescent light from the tissue which passes through the other half of the objective. The fluorescent light falls on to the cathode of a photomultiplier whose output is amplified by a lock-in amplifier. Scanning across the cornea is achieved by a stepper motor coupled to the fine focus of the microscope. The performance of the instrument has been assessed by studying the transport of fluorescein, carboxyfluorescein, and rhodamine B through intact rabbit corneas. A depth resolution of about 10 mu m has been achieved with a 40*objective. This resolution is sufficient to determine concentration gradients in the epithelium as well as the stroma and to partially resolve the endothelium. The potential errors in the technique are discussed.