In Vivo Confocal Microscopy of the Cornea: New Developments in Image Acquisition, Reconstruction, and Analysis Using...

The optical sectioning ability of confocal microscopy allows high magnification images to be obtained from different depths within a thick tissue specimen and is thus ideally suited to the study of intact tissue in living subjects. In vivo confocal microscopy has been used in a variety of corneal research and clinical applications since its development over 25 years ago. In this article we review the latest developments in quantitative corneal imaging with the Heidelberg Retinal Tomograph with Rostock Corneal Module (HRT-RCM). We provide an overview of the unique strengths and weaknesses of the HRT-RCM. We discuss techniques for performing 3-D imaging with the HRT-RCM, including hardware and software modifications that allow full-thickness confocal microscopy through-focusing (CMTF) of the cornea, which can provide quantitative measurements of corneal sublayer thicknesses, stromal cell and extracellular matrix backscatter, and depth-dependent changes in corneal keratocyte density. We also review current approaches for quantitative imaging of the subbasal nerve plexus, which require a combination of advanced image acquisition and analysis procedures, including wide-field mapping and 3-D reconstruction of nerve structures. The development of new hardware, software, and acquisition techniques continues to expand the number of applications of the HRT-RCM for quantitative in vivo corneal imaging at the cellular level. Knowledge of these rapidly evolving strategies should benefit corneal clinicians and basic scientists alike.