Calibration of measurements in vivo of fluorescein in the cornea

Quenching of fluorescence of fluorescein is not observed with broad field fluorophotometers. Fluorophotometric equipment which measures the fluorescence in a tiny spot has, however, been reported to underestimate the molarity of fluorescein in the rabbit corneal stroma by as much as a factor of two. In this experiment, quenching was measured in the rabbit cornea with two scanning fluorophotometers. The quenching was measured by four different techniques: (1) by elution of fluorescein, (2) by elution of albumin, (3) by polarization of fluorescence, and (4) by spectrofluorophotometry. It was estimated by all four methods that quenching in the living rabbit cornea with these instruments is approximately 20%. Taken together, the four experiments suggest that the quenching of fluorescence of fluorescein can be explained entirely on the basis of the interaction of fluorescein and albumin in the stroma.