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 (