Endothelial cell death above photo-disrupted (PD) and non-photo-disrupted (A) Bright field stereomicroscopy image of the cornea containing both the photo-disrupted and non-photo-disrupted sections.

<p>From the image, photo-disrupted and non-photo-disrupted areas can not be identified. (B) Dark field image of the same section of the cornea where focus was adjusted to observe the presence of the photo-disrupted area. (C) The ratio of healthy cell areas to total area analyzed is plotted in the region where we performed the laser treatment at 50 µm depth and an equally big region beside. No significant difference was observed between the two ratios (p=0.34, <i>t</i>-test). The data was tested for normality using Shapiro-Wilk test and was found to be with normal distribution. </p

Photo-disruption depth accuracy.

<p>(A) The photo-disruption was set at 50 µm from the endothelial surface and the mean (±standard deviation) measured distance was 50±3 µm throughout the 9 mm dissection (B) This micrograph illustrates the ability to vary the photo-disruption depth along the cornea. From left to right, the photo-disruption depth was set at 50 µm, 100 µm, 150 µm, 200 µm from the endothelial surface. </p

Endothelial cell death as a function of photo-disruption depth.

<p>Planar cuts in which we intentionally focused the laser within the endothelial cell layer in the center of cornea were performed. Due to the curvature of the cornea and the planar cut performed, the depth of the photo-disruption was minimum at the center (where cell death is observed) and increased towards the periphery (where no cell death is observed). After dissection, corneas were stained with trypan blue and alizarin red S and photographed. The ratio of the number of pixels in a region of interest that belong to healthy cells to the total number of pixels is plotted. The colored symbols represent the data for six different samples and their average represented by black solid line. The healthy ratio increased to 50% at a photo-disruption depth of 27 µm and to more than 99% at a depth of 34 µm.</p

Schematic of the optical system used for corneal dissection and imaging.

<p>The abbreviated components are: movable lens (ML), polarization maintaining fiber (PMF), collimator (C1, C2), transmission diffraction grating (TDG), achromatic lens (ACL), wedge beam splitter and charged coupled device camera (CCD camera) .</p