Dynamic molecular resolution imaging of preocular fluid impressions

Abstract

Aim: The preocular fluid is renewed with molecules secreted by the underlying cells and with lacrimal gland secretions, while maintaining a stable surface topography. The authors tested the hypothesis that interactions between gelled and newly inserted mucins are the key to this stability.

Methods: Using atomic force microscopy, the authors studied the topography of the freshly isolated preocular fluid obtained by impression cytology. The effects of adding mucins to this impression were compared with adding mucins to a pure mucin macromolecular assembly as a single component control to the more complex preocular fluid. The control structure was built up by repeated addition of pure ocular mucin to a tethering surface.

Results: Imaging at molecular resolution showed a thin layer of superficial preocular fluid with an appearance consistent with a gel that was very flat, with surface roughness of approximately 0.1 nm. Mucin molecules adhering to a clean flat surface maintained their individual character when overlapping, whereas molecules integrating in the impression could not be followed individually. Both the preocular impression and the pure mucin assembly were stable under imaging for at least 90 minutes. The roughness of the pure mucin network decreased as more mucin was added. In contrast, there was a small increase in the roughness of the 2.25 μm² area of impression over the 60 minutes of continuous imaging, although locally there appeared to be infill of low height features. Disulphide bond breaking resulted in the collapse of the imaged structure in both the pure mucin control and the more complex ex vivo preocular impression.

Conclusions: Polymeric mucins linked by disulphide bonds prevent or lessen loss of ocular surface material into the surrounding aqueous tears.