June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Investigation of conventional and transepithelial riboflavin/UVA corneal crosslinking by nonlinear optical microscopy
Author Affiliations & Notes
  • Giuseppe Lombardo
    IPCF - Unit Support of Cosenza, CNR, Rende (Cosenza), Italy
  • Norberto Liborio Micali
    CNR - IPCF, Messina, Italy
  • Valentina Villari
    CNR - IPCF, Messina, Italy
  • Dario Rusciano
    Sooft SPA, Montegiorgio, Italy
  • Marco Lombardo
    IRCCS Fondazione Bietti, Rome, Italy
  • Footnotes
    Commercial Relationships Giuseppe Lombardo, None; Norberto Liborio Micali, None; Valentina Villari, None; Dario Rusciano, SOOFT SPA (E); Marco Lombardo, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3366. doi:
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      Giuseppe Lombardo, Norberto Liborio Micali, Valentina Villari, Dario Rusciano, Marco Lombardo; Investigation of conventional and transepithelial riboflavin/UVA corneal crosslinking by nonlinear optical microscopy. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3366.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: To investigate the effect of conventional and transepithelial corneal crosslinking in human donor corneal tissues using two-photon optical microscopy.

Methods: Eight human donor corneas were imaged using two-photon microscopy. The corneas were separated in two groups: 1) tissues without epithelium that were treated by conventional corneal crosslinking and 2) tissues with intact epithelium that were treated by a novel transepithelial corneal crosslinking. After soaking with riboflavin 0.1% solutions, all tissues were irradiated with a 10 mW UVA lamp for 9 minutes. The 810 nm femtosecond laser was used to perform two-photon fluorescence (TPF), forward- and back-second harmonic generation (F-SHG and B-SHG) axial scanning measurements in each tissue. TPF and SHG images were used for analysis of stromal concentration of riboflavin, that was determined after normalizing the fluorescence intensity by the B-SHG signals.

Results: High-quality images were obtained before and after conventional and transepithelial crosslinking in all samples. The stromal concentration of riboflavin was on average 0.015% after transepithelial corneal soaking. Although the stromal diffusion of the novel transepithelial riboflavin solution varied between samples, the results were comparable with those obtained after stromal soaking with 20% dextran-enriched riboflavin solution (0.011%). After crosslinking, stromal concentration of riboflavin was on average 43% and 46% lower than baseline measurements after transepithelial and conventional UVA irradiation respectively. The SHG signals increased both after transepithelial and conventional corneal crosslinking procedures.

Conclusions: Two-photon optical microscopy signals provide relevant information to investigate the transepithelial diffusion and stromal concentration of novel riboflavin ophthalmic solutions. The technique is reliable to assist with the development of enhanced transepithelial ophthalmic solution and with the improvement of clinical outcome following corneal crosslinking.


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