Corneas stored in organ culture at 31°C in medium (CorneaMax [Eurobio, Les Ulys, France] or Inosol [Baush and Lomb Chauvin Opsia, Labége, France]) were studied before the deswelling process. The endothelial surface was incubated with 0.9% sodium chloride (Aguettant, Lyon, France) for 4 minutes for visualization of the cell borders by a process of gradual dilation of the intercellular spaces. This is a dynamic and transient phenomenon limited by an equilibration between the intracellular and the extracellular spaces, after which cell borders again become less obvious. Endothelium was viewed through a long working distance 10× objective under a direct optical microscope (DMLB; Leica Microsystems GmbH, Wetzlar, Germany). When cell borders were optimally discernible, endothelial photographs were acquired using a monochrome CCD video camera (XC-ST50CE; Sony, Tokyo, Japan) installed on the microscope and digitized using a video frame grabber (DT-3155; Data Translation, Marlboro, MA). Three wide-field (1000 μm × 750 μm) images of three randomly chosen nonadjacent zones of the endothelium contained within the central 8-mm diameter were taken at a resolution of 768 × 576 pixels in 8-bit gray level and saved in bitmap (BMP) format. Image quality was graded by two observers on a three-level score, depending on the visualization of cell borders, background noise, and surface area of the image with visible ECs.
12 The score was deemed good if the cell borders were very well visualized, if the background noise was low or nonexistent, and if the cells were visible on two thirds or more of the image area. It was deemed average if visualization of the cell borders was good, if the background noise was moderate, and if the cells were visible on one third to two thirds of the image area. It was deemed poor if the cell borders were difficult or impossible to visualize, if the background noise was high, and if cell borders were visible on less than one third of the image area. Thirty corneas with images of different scores (good, 12 [40%]; average, 9 [30%]; poor, 9 [30%]) were chosen for the study so as to represent the routine eye bank practice. The sample size was chosen as a balance between the feasibility of involving multiple technicians of two eye banks in a research procedure conducted in addition to their routine clinical activities and the minimum number required for a valid statistical calculation. High-quality laser printouts of the images on premium matte photograph paper (HP Q6549A; Hewlett-Packard, Geneva, Switzerland) were obtained at resolution of 600 dpi in A4 format (Laser Shot LBP-1120; Canon Europe, Uxbridge, UK) for manual counting purposes. A certified micrometer (Leitz GmbH, Postfach, Germany) was also photographed in the same conditions and served as a reference scale for the endothelial images. A grid overlay simulating the microscope reticule, with each vertical column designated from A to H and each horizontal line from 1 to 6, was designed with software (Photoshop version 7.0.1; Adobe Inc., San Jose, CA) and printed on A4-sized transparency sheets (Epson, Long Beach, CA) using the same printer. The dimensions of the grid were similar to the visual appearance of the grid placed in the eyepiece—the square of 120 μm × 120 μm (external dimension), with a line thickness of 2.6 μm—taking account the 10× objective and the internal magnification factor of that microscope.