Journal
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
Volume 57, Issue 2, Pages 476-483Publisher
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.15-18651
Keywords
cross-linking; riboflavin; two-photon optical microscopy; spectrophotometry
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Funding
- National Framework Program for Research and Innovation PON [01_00110]
- Italian Ministry of Health
- Fondazione Roma
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PURPOSE. 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/cm(2) 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 mu m; the concentration of riboflavin was almost constant up to 320 6 53 mu m 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.
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