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H B Chen, S Yamabayashi, B Ou, Y Tanaka, S Ohno, S Tsukahara; Structure and composition of rat precorneal tear film. A study by an in vivo cryofixation.. Invest. Ophthalmol. Vis. Sci. 1997;38(2):381-387.
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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.
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