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Daniel M Gore, Anca Margineanu, Paul French, David O'Brart, Chris Dunsby, Bruce D S Allan; Two-photon fluorescence (TPF) microscopy of corneal riboflavin absorption. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4215.
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© ARVO (1962-2015); The Authors (2016-present)
To correct for attenuation in TPF measurements of riboflavin absorption in porcine corneas
A 300µl reservoir of 0.1% riboflavin was placed (air-bubble free) on top of epithelial-debrided porcine corneas (globe intact). TPF imaging was performed through this reservoir with 10µm z-stacks across the cornea repeated every 2.5 minutes (maximum 60 minutes). Riboflavin was excited by two-photon light of 890nm wavelength, with fluorescence signal detected between 525-650nm. TPF signal attenuation was corrected with reference to a uniformly soaked plot from each eye towards the end of the 60 minute soak. This protocol was repeated in 4 eyes. In selected eyes, z-stacks were additionally performed through uniformly-soaked excised corneal buttons.
Significant TPF signal attenuation was observed in all eyes, with the signal decreasing linearly with depth in uniformly soaked tissue. This signal loss was observed for intact globes and when imaging through excised corneal buttons (epithelial-debrided surface up or down). Cross-sectional TPF images taken of additional excised corneal strips confirmed the tissue was uniformly soaked so that the decrease in signal was not due to variations in riboflavin concentration. After correcting for this signal attenuation, we observed TPF signals increased with longer riboflavin soak duration, with the maximum tissue concentration recorded at 0.06%. 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 0.053% (0.002, standard deviation) was achieved at a depth of 300µm. Half this concentration (0.026%) was observed within the first 5 minutes.
TPF microscopy has previously been applied to study corneal riboflavin absorption without accounting for depth-dependent signal attenuation (Cui et al. IOVS 2011;52:2556-64). Our data suggest this signal loss needs to be taken into account and we report a method of correcting the TPF measurements to more accurately quantify the concentration of riboflavin throughout the cornea. The minimum concentration for effective cross-linking is unknown: Our data suggest this may be effected with a tissue concentration of approximately 0.05% within the 250-350µm demarcation zone commonly seem after treatment.
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