Two-Photon Fluorescence Microscopy for Determination of the Riboflavin Concentration in the Anterior Corneal Stroma When Using the Dresden Protocol

Abstract

Citation: Seiler TG, Ehmke T, Fischinger I, et al. Two-photon fluorescence microscopy for determination of the riboflavin concentration in the anterior corneal stroma when using the Dresden protocol. Invest Ophthalmol Vis Sci. 2015;56:6740-6746. DOI:10.1167/iovs.15-17656 PURPOSE. To determine the riboflavin concentration gradient in the anterior corneal stroma when using the Dresden protocol with different dextran solutions. METHODS.Three different groups of porcine corneas, five each, were compared regarding the riboflavin concentration in the anterior stroma. Before all experiments, stable hydration conditions were established for the corresponding solution. All groups were treated with 0.1% riboflavin in different dextran solutions (15%, 16%, 20%). After imbibition, two-photon microscopy was used to determine fluorescence intensity. For signal attenuation and concentration determination corneas were saturated and measured a second time by twophoton microscopy. Additionally, the distribution was calculated mathematically and compared to the empiric results.RESULTS. Riboflavin concentration is decreasing with depth for all dextran solutions. A nearly constant concentration could be determined over the first 75 lm. Analysis of the fit functions leads to diffusion coefficients of D ¼ 2.97 3 10 À7 cm 2 /s for the 15% dextran solution, D ¼ 2.34 3 10 À7 cm 2 /s for the 16% dextran solution, and D ¼ 1.28 3 10 À7 cm 2 /s for the 20% dextran solution.The riboflavin gradients of the 20% dextran group were statistically significantly different from 15% dextran starting at a depth of 220 lm and deeper (P ¼ 0.047). The 16% dextran group differed statistically at a depth of 250 lm and deeper (P ¼ 0.047). These results show a significant difference to those published previously. CONCLUSIONS.With correct settings two-photon microscopy is a precise way to determine the concentration of riboflavin in cornea. The measured gradient is excellently fit by a Gaussian distribution, which comes out as a solution of Fick's second law.

Riboflavin Concentrations at the Endothelium During Corneal Cross-Linking in Humans

“…Afterward, remnants of epithelium were removed using a blunt hockey knife. As previously described, 19 a reservoir containing 15% dextran T-500 solution was placed on the anterior side of the cornea to achieve stable physiological hydration via both surfaces of the cornea. After a stable hydration state had been reached (three identical consecutive pachymetries, 10 minutes apart) the first twophoton microscopy scan was performed.…”

“…As corneal thickness was maintained constant throughout the entire course of the experiment, this landmark was used for the determination of absolute riboflavin concentration, which was performed as previously described. 19 In brief, obtained mean gray values of each corneal depth for a predetermined region of interest (ROI) were compared to those of the same corneal depth in corresponding corneal stacks (after 10 minutes of riboflavin imbibition and after riboflavin saturation) calibrating the signal attenuation due to absorption and scattering effects within the cornea.…”

Loss of Tryptophan Fluorescence Correlates With Mechanical Stiffness Following Photo-Crosslinking Treatment of Rabbit Cornea

“…Previous studies have shown a consistent concentration of riboflavin in the corneal stroma up to 100 lm. 10 Riboflavin acts as both a photosensitizer in the stroma and an absorber of UV light, protecting the underlying ocular structures from UV damage. 7 The exact mechanism by which photo-crosslinking strengthens the cornea has not been elucidated, but a prevailing theory is that riboflavin creates reactive oxygen species within the corneal stroma, which interact with proteins present in the collagen fibrils to produce crosslinks.…”

Chlorin e6 mediated photodynamic inactivation for multidrug resistant Pseudomonas aeruginosa keratitis in mice in vivo

“…Taking into account the insufficient diffusion of this photosensitizer into the corneal stroma51, the molar concentration of 0.005% MB used in regimen III was 156 μM, and thus slightly higher than the 100 μM MB recommended in a previous in vitro study49, but just equivalent to 0.009% Ce6. Moreover, for the same amount of substance, the ROS generated from irradiated MB is just about 80% of that of Ce637.…”