title: All-Optical Method to Assess Stromal Concentration of Riboflavin in Conventional and Accelerated UV-A Irradiation of the Human Cornea.
abstract: OBJECTIVE
  We investigated the concentration of riboflavin in human donor corneas during corneal cross-linking using two-photon optical microscopy and spectrophotometry.
  
  
  METHODS
  Eight corneal tissues were de-epithelialized and soaked with 20% dextran-enriched 0.1% riboflavin solution for 30 minutes. After stromal soaking, three tissues were irradiated using a 3 mW/cm2 UV-A device for 30 minutes and three tissues irradiated using a 10 mW/cm2 device for 9 minutes. Two additional tissues were used as positive controls. A Ti:sapphire laser at 810 nm was used to perform two-photon emission fluorescence (TPEF) and second harmonic generation axial scanning measurements in all specimens before and after stromal soaking and after UV-A irradiation. In addition, spectrophotometry was used to collect the absorbance spectra of each tissue at the same time intervals. Analysis of the absorbance spectra and TPEF signals provided measures of the concentration depth profile of riboflavin in corneal stroma.
  
  
  RESULTS
  After stromal soaking, the average peak concentration of riboflavin (0.020% ± 0.001%) was found between a stromal depth of 100 and 250 μm; the concentration of riboflavin was almost constant up to 320 ± 53 μm depth, then decreased toward the endothelium, though riboflavin was still enriched in the posterior stroma (0.016%% ± 0.001%). After conventional and accelerated UV-A irradiation, the concentration of riboflavin decreased uniformly 87% ± 2% and 67% ± 3% (P < 0.001), respectively.
  
  
  CONCLUSIONS
  The combined use of two-photon optical microscopy and spectrophotometry provides relevant information for investigating the concentration of riboflavin in corneal stroma. The method can assist with the assessment of novel riboflavin formulations and different UV-A irradiation protocols.
authors: Consiglio Nazionale delle Ricerche Istituto per i Processi Chimico-Fisici, CNR-IPCF, Messina, Italy 2Vision Engineering Italy srl, Rome, Italy.
authors: Giuseppe
authors: G
authors: Lombardo
authors: Consiglio Nazionale delle Ricerche Istituto per i Processi Chimico-Fisici, CNR-IPCF, Messina, Italy.
authors: Norberto Liborio
authors: NL
authors: Micali
authors: Consiglio Nazionale delle Ricerche Istituto per i Processi Chimico-Fisici, CNR-IPCF, Messina, Italy.
authors: Valentina
authors: V
authors: Villari
authors: Fondazione G.B. Bietti IRCCS, Rome, Italy.
authors: Sebastiano
authors: S
authors: Serrao
authors: Fondazione G.B. Bietti IRCCS, Rome, Italy.
authors: Marco
authors: M
authors: Lombardo
mesh_terms: D000368:Aged; D003094:Collagen / Q000378:metabolism; D003315:Cornea / Q000528:radiation effects; D003319:Corneal Stroma / Q000378:metabolism*; D003432:Cross-Linking Reagents; D006801:Humans; D036641:Microscopy, Fluorescence, Multiphoton; D008875:Middle Aged; D061848:Optical Imaging; D017319:Photosensitizing Agents / Q000493:pharmacokinetics*; D012256:Riboflavin / Q000493:pharmacokinetics*; D013053:Spectrophotometry; D014019:Tissue Donors; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D003094:Collagen; D012256:Riboflavin