Seiler, Günter Theodor MichaelGünter Theodor MichaelSeilerBatista, AnaAnaBatistaFrüh Epstein, BeatriceBeatriceFrüh EpsteinKoenig, KarstenKarstenKoenig2024-10-282024-10-282019-05-01https://boris-portal.unibe.ch/handle/20.500.12422/181054Purpose To determine the riboflavin concentration in the posterior corneal stroma, Descemet's membrane, and endothelium prior to UV irradiation in corneal cross-linking (CXL) in humans. Methods Five human deepithelialized cadaver corneas were mounted into artificial anterior chambers. After the establishment of stable physiological hydration, two-photon imaging with a certified multiphoton tomograph was used to determine fluorescence intensity and second harmonic generation signals from collagen throughout each cornea by optical sectioning, with a step size of 2.5 μm. Afterward, 0.1% riboflavin solution was applied to the anterior corneal surface, similar to the standard CXL protocol. To determine the absolute riboflavin concentration immediately before UV irradiation, corneas were measured by two-photon imaging just at the end of the riboflavin imbibition and after riboflavin saturation. Results The topical application of 0.1% riboflavin results in a riboflavin concentration that decreases to 0.035% in the posterior stroma. Inside Descemet's membrane and endothelium, the concentration drops further to only approximately 0.015% at the endothelial level. Local riboflavin distribution indicates a predominantly paracellular passive diffusion of riboflavin into the anterior chamber. Conclusion The experimentally determined riboflavin concentration of 0.015% at the endothelium shows a substantial discrepancy of a factor of 1.7 to the previously theoretically calculated 0.025%. A lower riboflavin concentration at the endothelium may enable higher radiant exposures and further improve the efficacy of CXL.en600 - Technology::610 - Medicine & healtharticle10.7892/boris.1317083109983010.1167/iovs.19-26686