Three‐dimensional imaging of corneal cells using in vivo confocal microscopy

Confocal microscopy is a unique and powerful imaging paradigm which allows optical sectioning through intact tissue. Real‐time tandem scanning confocal microscopy has previously been used to generate high‐magnification two‐dimensional (2‐D) images of cells in living organ systems. Inherent problems with movement, however, have prevented the in vivo acquisition of complete 3‐D datasets. The development of a new objective lens, used in combination with specialized real‐time image acquisition procedures, has allowed sequential serial sections to be obtained in vivo from the rabbit cornea for the first time. These sections can be digitially registered and stacked on the computer to provide a 3‐D reconstruction of the corneal cells. This technique should serve as a useful method for studying 3‐D structures and analysing 4‐D phenomena at the cellular level in living animals. Three‐dimensional images of a stromal nerve in normal rabbit cornea and of fibroblasts within a rabbit corneal wound are presented as examples of current capabilities.