Structure and composition of rat precorneal tear film. A study by an in vivo cryofixation.

PURPOSE To visualize the in vivo structure and to investigate the composition of rat precorneal tear film. METHODS An in vivo cryofixation with freeze substitution method of electron microscopy was used for the study. For light and transmission electron microscopy, a small amount of aluminum powder was used as a tracer spread on the corneal surface. The eyeballs were immediately and quickly frozen by pouring an isopentane-propane mixture cooled by liquid nitrogen directly over the eyes. For scanning electron microscopy, the corneal surface was freeze-fractured after the cryofixation. The specimens were then freeze-substituted and prepared conventionally for microscopic observation. RESULTS The tear film appeared as a layer of homogeneous and fine network-like structures varying from 2 to 6 microns in thickness on the corneal surface, with a membrane-like layer covering its surface. The aluminum powder was located on the surface of the tear film. The tear film could be removed completely by applying 10% or 20% acetylcysteine, but not by phosphate buffer. CONCLUSIONS The in vivo structure of the rat tear film is composed primarily of mucus, with a lipid layer covering its surface but without a free aqueous layer. The "three layers theory" of tear film structure requires revisions.