Examination of Murine Tear Film

To define spatially any free aqueous layer in murine tear film. A pre-zeroed microelectrode was touched to the superficial corneal epithelium and then raised in steps of 1 micro m through the murine tear film into the air and then retraced along the same path. Other murine tear films were partially probed with a spatial resolution of 0.1 micro m. The reference microelectrode was placed in a fragment of 3% polyacrylamide gel equilibrated against 154 mM NaCl and located on the nasal quadrant of the scleral conjunctiva. Other murine corneas were quick frozen in melting isopentane and freeze substituted or pretreated with cetylpyridinium chloride and then examined by transmission electron microscopy. The recorded electrical profiles of the tear film were reproducible in each preparation and showed a relatively uniform positive electrical potential throughout their whole thickness, except within 0.5 micro m of the epithelial surface when the potential reversed to negative values. The thickness of mouse tear film averaged 7.4 +/- 0.8 micro m (mean +/- SD, n = 40). The electron microscope images showed the murine tear film to have a relatively uniform positive electron density throughout the thickness. Electrical profiles of the murine tear film presented no evidence of a separate free aqueous phase. The tear film is observed as an aqueous gel that includes anion-exchanging polyelectrolytes throughout most of its thickness, but within 0.5 micro m of the epithelial surface, it changes to cation-exchanging polyelectrolytes. Electron microscope images provide some supporting evidence.