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Pavel Kamaev, Radha Pertaub, Marc Friedman, David Muller; Implications of New Absorption and Fluorescence Measurements of Riboflavin for Corneal Cross-linking. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6805.
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To determine the absorption and fluorescence of riboflavin (RF) as a function of concentration and determine its implications for corneal cross-linking.
An Evolution 300 spectrophotometer (Fisher Scientific) utilizing a micro-cuvette (Agilent Technologies) with a 0.5mm cell length was used to measure UVA (370 nm) absorption of RF at varying concentrations up to ~0.20% w/v to calculate the absorption coefficient of riboflavin (σRF). A UVA LED source illuminates a digital micromirror device (DMD) which then images a straight vertical slit onto a microscope slide with coverslip. A camera (Basler acA1300-30gm) mounted with a long-pass filter is placed at a 45° angle to the slide. The camera detects the fluorescence emission through the cross-section of the RF. The fluorescence of RF was measured at varying concentrations up to 0.225 % w/v.
The absorption spectrum of RF is linear as predicted by Beer’s law to at least a concentration of 0.20%: σRF=235x[RF, % w/v], cm-1. The fluorescence emission in our setup was determined to be linear as well which verifies our absorption measurements: Pixel Intensity = 1157x[RF, % w/v].
The safety profile of corneal cross-linking with RF was previously based upon the absorption spectra of RF determined by Spoerl et al.1 to have saturated at 0.04% which we believe was a function of their measurement technique. The implications of RF absorption being linear predict that more absorption will occur at the anterior cornea. This suggests a better safety profile of corneal cross-linking with RF and much less absorption of the cornea than previously thought. This linear absorption and fluorescence profile also lends itself to the potential measurement of RF concentration in the cornea in real time.1 Spoerl et al., "Safety of UVA-Riboflavin Cross-Linking of the Cornea". Cornea: v.26, p.385-389. 2007.
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