Two-photon imaging of the cornea visualized in the living mouse using vital dyes

To acquire morphological and component information on the overall cornea in the living C57BL/6 mouse using fluorescent viability dyes and two-photon (2PH) laser microscopy.

Corneas were scanned using a 2PH laser scanning fluorescence microscope after staining with plasma membrane stain and Hoechst 33342. Representative 2PH images of corneal cells were analyzed and restructured using Imaris software.

With the plasma membrane- and cell-permeant nuclear counter live cell fluorescent probe, the morphology of corneal cells and the construction of cornea were observed clearly. The general in vivo morphology of the cornea clearly showed three different cellular layers, two interfaces, and the nerve fibers. Our study detailed all of the corneal cells with clear cell nuclei and cell boundaries at the sections parallel to the surface of the cornea. Moreover, cellular stereoscopic images, the relationships of the neighbor cells, and the interfaces of different layers were also displayed distinctly with three-dimensional construction of the 2PH imaging. Our research showed for the first time that there are three or four layers of epithelial cells and seven or eight layers of keratocytes in mice.

Our study clearly showed the anterior limiting lamina and Descemet's membrane in the mouse cornea and showed for the first time that there are three or four layers of cells in the epithelium and seven or eight layers of keratocytes in the stroma of mice. These results are necessary primarily to contribute important insights into the anatomy and pathology of the cornea in mice.