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L. J. Nagy, L. Ding, T. Bubel, J. Swanton, W. H. Knox, K. R. Huxlin; Potentiating Femtosecond Intra-Tissue Refractive Index Shaping (IRIS) in the Living Cornea With Sodium Fluorescein Doping. Invest. Ophthalmol. Vis. Sci. 2009;50(13):568.
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© ARVO (1962-2015); The Authors (2016-present)
To test the hypothesis that intra-tissue refractive index shaping (IRIS) can be successfully performed in living corneas, and that sodium fluorescein (Na-Fl), an enhancer of two-photon absorption, increases the scanning speed and refractive index (RI) change attainable in a dose-dependent, non-toxic manner.
3 corneas from 2 adult domestic short hair cats were removed immediately upon death. Each cornea was cut into 6 pieces, each placed in chilled Optisol GS (Bausch & Lomb Inc.) or Optisol containing 0.25%, 0.5%, 1%, 1.5% or 2.5% Na-Fl for 2 hrs. An 800nm Ti:Sapphire femtosecond laser with 100fs pulse duration, 80MHz repetition rate and 120mW average power was used to perform IRIS in each piece, creating multiple sets of 6 line gratings. The lines were 1µm thick, 10µm apart and 100µm below the tissue surface. After IRIS, the RI change of gratings created at different speeds (0.1-, 0.5-, 1-, 2-, 5mm/s) was measured using a calibrated differential interference contrast microscope. The tissue was then stored in Optisol for 4 hrs, fixed in 1% paraformaldehyde in 0.1M PBS for 10 minutes, cryoprotected and sectioned on a cryostat before undergoing TUNEL staining (Apoptag Peroxidase In Situ Apoptosis Detection Kit, Millipore Corp.).
The scanning speed and the magnitude of RI change attained in living corneas increased as a function of Na-Fl doping concentration. In undoped corneas, scanning at 0.1mm/s increased the RI of the tissue by 0.005. In doped corneas, RI changes as large as 0.02 were measured. This was attained in 0.5% doped tissue by scanning at 0.5-1mm/s, in 1% doped tissue by scanning at 1mm/s and in 2.5% doped tissue by scanning at 5mm/s. TUNEL staining showed no evidence of cell death in or around any of the grating lines 4 hrs post-IRIS.
Na-Fl increased the scanning speed and RI change attainable in fresh corneal tissue during IRIS in a dose-dependent manner and without causing immediate cell death. Ongoing studies are exploring the possibility of using IRIS to alter the optical properties of corneal tissue in situ over an extended period of time.
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