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M.L. Denton, K. Schuster, L. Estlack, G. Noojin, B. Rockwell; Near–IR Laser–Induced Oxidative Stress in RPE Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1592.
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Purpose: Techniques utilizing mode–locked near–IR (NIR) laser beams are becoming increasingly popular. Safety of patient and clinician is of great importance considering that the typical laser wavelengths used in these applications are outside of the visible spectrum. Our goal is to identify threshold power densities of mode–locked lasers required to generate photochemical oxidation. Methods: Cultures of hTERT–RPE1 cells were exposed to a laser running at 810 nm while being imaged in real–time using a confocal microscope. Beam diameter and power delivered were easily measured by conventional methods because the beam was not passed through the microscope objective. Using fluorescent indicator dyes, dose–dependent measures of oxidation were documented and a possible subcellular origin for the oxidation identified. Each time–lapse series included a pre–laser exposure image as a control for quantification. Results: NIR laser exposure (mode–locked but not CW) generated oxidation in hTERT–RPE1 cells as indicated by three different fluorescent dyes (CM–H2DCFDA, dihydroethidium, and CM–H2XROS). Oxidation was found in the cytoplasm, nucleus and mitochondria of the cells. Fluorescence intensity per unit time (using CM–H2DCFDA) increased in a dose–dependent manner and the rates of oxidation from various laser irradiances were used to estimate a threshold of 3000 W/cm2 (SD 400) under standard conditions, corresponding to peak irradiances of about 4 x 108 W/cm2 (90 fsec, 80 MHz mode–locked). When considering the optics of the eye, this would correspond to about 10 mW TIE. Ascorbic acid inhibited the overall oxidation to 17% of control (P<0.001), leaving only mitochondrial contributions. N–acetyl–L–cysteine was much less effective at inhibiting the oxidation (63% of control). Conclusions: Photochemical oxidation by mode–locked NIR lasers occurs at relatively low power densities, and appears to originate in mitochondria.
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