A prototype two-detector confocal microscope for in vivo corneal imaging

Confocal microscopy through-focusing (CMTF) of the cornea produces a three-dimensional (3-D) display of corneal structure and intensity profiles that allow objective measurements of corneal sublayer thickness and relative assessment backscattering of light. In this study, a prototype confocal instrument was evaluated in which a photon counting photomultiplier tube (PMT) detector was added to provide faster and more quantitative measurements, while still maintaining the imaging capability of the microscope. To acquire images and measure backscattered light simultaneously, an uncoated pellicle beam splitter was incorporated into the light path of the confocal microscope. This beam splitter reflects 8% of the confocal signal to the PMT. The CMTF scans were performed on four rabbits using the prototype instrument. Corneal images and 3-D reconstructions acquired with and without the beam splitter in the light path appeared identical. Both the camera and PMT CMTF curves had easily identifiable peaks corresponding to the epithelium, basal lamina, and endothelium. No significant differences were found between PMT and camera CMTF measurements of epithelial, stromal, or corneal thickness (n = 12 scans). Furthermore, a high correlation was found between camera and PMT measurements (linear regression analysis, y = 0.999 x -0.4, r = 0.99, p < 0.001). The data suggest that by adding a pellicle beam splitter, CMTF intensity data can be acquired using a PMT. The PMT has a faster sampling rate and greater dynamic range than the camera and provides a count of the photons detected. Thus, the instrument has the potential for improving corneal pachymetry and back-scattering measurements while still providing high-resolution corneal images.