A microfluorometer for measuring diffusion of fluorophores across the cornea

A microscope has been modified to 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 microns has been achieved with a 40x 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, resulting from limited resolution, light scattering and absorption, quenching of fluorescence, and binding of the fluorophores, are discussed.