Fluorescence Depth Measurements of Rose Bengal Diffused into Cornea and pHEMA measured by Two-photon Fluorescence Microscopy

Abstract

Purpose : To evaluate the diffusion of Rose Bengal (RB) into corneas and pHEMA, a hydrophilic polymer, in order to understand its limited penetration into cornea when used as a photosensitizer for cornea crosslinking.

Methods : RB solutions at 0.1% and 0.5% w/v were prepared in saline. Hydrated, unpolished blocks of hydrophilic polymer pHEMA were floated on RB solutions for 5, 20, and 60 seconds. The RB solutions were also applied to de-epithelialized corneas of porcine eyes (n=5; < 36 hours post-mortem) by adding eight drops over two minutes. The RB-stained pHEMA samples and corneas were placed in a custom two-photon fluorescence microscope (Spectra Physics Mai Tai femto-second laser, λ_excitation=745nm, λ_fluorescence=500-700nm, τ_pulse=80fs, P_ex=1-10mW; N.A. = 0.9 objective). Fluorescence levels were measured with a photomultiplier tube (Hamamatsu H10682-210) while a motorized stage moved the samples through a 1-mm range in 10-µm steps, with a dwell time of 5s.

Results : The average ratios of maximum fluorescence intensities for the 0.5% to the 0.1% RB solutions found at the surface of the pHEMA blocks were 5.333±0.011, 2.333±0.004, and 1.636±0.002 for the 5, 20, and 60s samples, respectively. In pHEMA, 69.0±0.1% (for 0.1% RB) and 73.3±0.1% (for 0.5% RB) of the RB was found within the first 100mm, as determined from the area under the fluorescence z-scan curves. For RB-stained corneas, the average ratio of maximum fluorescence intensities was 0.598±0.001 for the 0.5% to the 0.1% RB solutions. Z-scans recorded 73.4±0.1% of the fluorescence signal in the first 100mm of corneas treated with 0.1% RB solution and 79.7±0.7% for the 0.5% RB solution.

Conclusions : In pHEMA blocks, the maximum peak fluorescence intensity was always bigger for the 0.5% RB solution, but in corneas the 0.1% solution produced larger maximum fluorescence intensity. The fluorescence z-scans of both pHEMA and corneas show that the RB travels further into the sample when a more dilute solution is applied. This indicates that a simple diffusion model, i.e. a direct application of Fick’s Second Law, does not completely describe the method of transport of RB into corneas or polymers.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.