Two-photon fluorescence excitation microscopy to assess transscleral diffusional pathways in an isolated perfused bovine eye model

To assess the feasibility of using two-photon microscopy to study the pattern of diffusion through the sclera of a tracer (tazarotenic acid [TA]).

Polyvinyl alcohol films containing 1% tazarotenic acid (PVA-TA) were applied to the equatorial sclera of isolated perfused bovine eyes. Two-photon microscopy (TPM) was used to determine the lateral spread and depth of penetration of TA in the sclera over time. Protein and collagen binding were determined, and calibration standards were prepared by TPM imaging at 10 μm depth in scleral samples that had been immersed for 24 hours in solutions of TA of 0.7, 7.0, or 70 μg/mL.

TA was weakly bound to collagen and sclera (<55%) but strongly bound to plasma protein (95%). In perfused eyes, 50 minutes after PVA-TA application, peak fluorescence in the sclera was detected at a 210-μm depth. By 85 minutes after application of the PVA-TA film, fluorescence had disappeared from surface layers of the sclera and was at maximum at 250 to 290 μm. Penetration of the tracer followed the track of scleral collagen bundles rather than that of the proteoglycan ground substance between collagen bundles.

TPM can image in real time the progressive diffusion of TA from its source in a PVA-TA film applied to the equatorial sclera of the isolated perfused bovine eye and follow its subsequent penetration deeper into the sclera. The data suggest that lateral spread and deeper penetration of the test compound occurred along the course of scleral collagen bundles. Imaging was possible to a depth of 340 μm, the average thickness of the human equatorial sclera.