title: Two-Photon Fluorescence Microscopy for Determination of the Riboflavin Concentration in the Anterior Corneal Stroma When Using the Dresden Protocol.
abstract: OBJECTIVE
  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 two-photon 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 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.
  
  
  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.
authors.affiliation: Klinik und Poliklinik für Augenheilkunde, Technische Universität München, Munich, Germany 2Institut für Refraktive und Ophthalmo-Chirurgie (IROC), Zurich, Switzerland.
authors.forename: Theo G
authors.initials: TG
authors.lastname: Seiler
authors.affiliation: Laser Zentrum Hannover e.V., Hanover, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Ehmke
authors.affiliation: Klinik und Poliklinik für Augenheilkunde, Technische Universität München, Munich, Germany 2Institut für Refraktive und Ophthalmo-Chirurgie (IROC), Zurich, Switzerland.
authors.forename: Isaak
authors.initials: I
authors.lastname: Fischinger
authors.affiliation: Klinik und Poliklinik für Augenheilkunde, Technische Universität München, Munich, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Zapp
authors.affiliation: Department of Ophthalmology, Rostock University Medical Center, Rostock, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Stachs
authors.affiliation: Institut für Refraktive und Ophthalmo-Chirurgie (IROC), Zurich, Switzerland.
authors.forename: Theo
authors.initials: T
authors.lastname: Seiler
authors.affiliation: Laser Zentrum Hannover e.V., Hanover, Germany 5Institut für Quantenoptik, Leibniz Universität Hannover, Hanover, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Heisterkamp
mesh_terms: D000818:Animals; D000869:Anterior Eye Segment / Q000378:metabolism*; D003319:Corneal Stroma / Q000378:metabolism*; D003911:Dextrans; D036641:Microscopy, Fluorescence, Multiphoton; D009883:Ophthalmic Solutions; D017319:Photosensitizing Agents / Q000493:pharmacokinetics*; D012256:Riboflavin / Q000493:pharmacokinetics*; D013552:Swine; D014018:Tissue Distribution
chemical_list: D003911:Dextrans; D009883:Ophthalmic Solutions; D017319:Photosensitizing Agents; D012256:Riboflavin