Purchase this article with an account.
B.E. Stuck, P.R. Edsall, K.P. Walker, III, S.T. Schuschereba, P.D. Bowman; Production of a Uniform Cellular Injury by Raster Scanning of Cells for the Study of Laser Bioeffects . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5515.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To produce a uniform response in cells to laser irradiation to better understand the biochemical and molecular basis of laser bioeffects.
ARPE–19 cells derived from human retinal pigment epithelium were cultured to 95% confluence in either 35 mm diameter glass bottom culture dishes or 24–well multiplates. A carbon dioxide laser (10.6 µm) with output at 2 W was used for irradiation. Cell viability was assessed by reduction of 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT assay), and heat shock protein 70 (hsp70) immunocytochemistry used at 6 h post–irradiation to assess the cellular response of viable cells. Initial attempts to produce a uniform or "tophat" exposure of the cells by selection of the central portion of the beam with a 2 mm circular aperture produced a non–uniform distribution of viable cells and hsp70 staining regardless of the distance of the aperture from the cell layer due to diffraction from the aperture. The intensity distribution of the beam was then measured with an automated beam scanning device confirming that the cellular response correlated with the actual intensity distribution. A computer–controlled raster scanning system was devised that produced uniformly viable cells that responded with a good hsp70 response. The velocity of the scanning 2.7 mm (1/e diameter) beam was 28.8 cm/s with an irradiance of 34.6 W/cm2.
Diffraction effects of 10.6 µm radiation through a 2 mm circular aperture abrogated attempts to produce a "tophat" intensity profile incident on the cells, as visualized both by immunostaining and cell viability, and hence a uniform injury to cells. However, cells exposed by a raster scanning of the beam were uniformly responsive across the monolayer as evidenced by hsp70 expression. The cellular response achieved with the scanning beam was comparable to that obtained by controlled dipping of the cells in 55 oC saline for 2s.
The cellular response to thermal injury from 10.6 µm was consistent with the intensity profile of the incident beam when the attempt to achieve a uniform intensity distribution by aperture selection was abrogated by diffraction effects. Exposure by raster scanning of the laser produced a uniform response across a cell monolayer. Sufficient numbers of cells are affected by this scanning procedure in an area of about 1 cm2 (about 50,000 cells) so that biochemical and molecular studies of the response and means to ameliorate it are now possible. This method of irradiation may also have application to in vivo studies of eye/laser interactions.
This PDF is available to Subscribers Only