Two-photon fluorescence microscopy of corneal riboflavin absorption.

PURPOSE: To correct for attenuation in two-photon fluorescence (TPF) measurements of riboflavin absorption in porcine corneas.
METHODS: Two-photon fluorescence imaging of riboflavin was performed using excitation at a wavelength of 890 nm, with fluorescence signal detected between 525 and 650 nm. TPF signal attenuation was demonstrated by imaging from either side of a uniformly soaked corneoscleral button. To overcome this attenuation, a reservoir of dextran-free 0.1% wt/vol riboflavin 5'-monophosphate in saline and hydroxypropyl methylcellulose (HPMC) was placed on top of porcine corneas (globe intact-epithelium removed). TPF imaging was performed through this reservoir with image stacks acquired at 10-μm steps through the cornea repeated at regular intervals for up to 60 minutes. A novel correction method was applied to achieve corneal riboflavin concentration measurements in whole eyes (n = 4).
RESULTS: Significant attenuation of the TPF signal was observed in all eyes, with the signal decreasing approximately linearly with depth in uniformly soaked tissue. Cross-sectional TPF images taken of excised corneal strips confirmed the tissue was uniformly soaked so that the decrease in signal was not due to spatial variations in riboflavin concentration. After correcting for signal attenuation, we observed increased riboflavin concentrations with longer soak duration, with the mean (standard deviation) maximum tissue concentration recorded at 0.094% (± 0.001) wt/vol [1.36 mg/mL]. Uniform riboflavin absorption was achieved after a minimum 50 minutes. Following a standard corneal cross-linking soak of 30 minutes, a mean stromal concentration of 0.086% (± 0.001) wt/vol [1.25 mg/mL] was achieved at a depth of 300 μm.
CONCLUSIONS: The accuracy of TPF measurements of corneal riboflavin absorption can be increased by applying a correction for depth-related signal attenuation.