Two-Photon Fluorescence Microscopy for Determination of the Riboflavin Concentration in the Anterior Corneal Stroma...

To determine the riboflavin concentration gradient in the anterior corneal stroma when using the Dresden protocol with different dextran solutions.

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 two-photon microscopy. Additionally, the distribution was calculated mathematically and compared to the empiric results.

Riboflavin concentration is decreasing with depth for all dextran solutions. A nearly constant concentration could be determined over the first 75 μm. Analysis of the fit functions leads to diffusion coefficients of D = 2.97 × 10-7 cm2/s for the 15% dextran solution, D = 2.34 × 10-7 cm2/s for the 16% dextran solution, and D = 1.28 × 10-7 cm2/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 μm and deeper (P = 0.047). The 16% dextran group differed statistically at a depth of 250 μm and deeper (P = 0.047). These results show a significant difference to those published previously.

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.