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(Investigative Ophthalmology and Visual Science. 2000;41:680-686.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

The Use of Atomic Force Microscopy for the Observation of Corneal Epithelium Surface

Miltiadis K. Tsilimbaris1, Eric Lesniewska2, Stella Lydataki1, Christian Le Grimellec3, Jean P. Goudonnet2 and Ioannis G. Pallikaris1

1 From the University of Crete Medical School, Department of Ophthalmology, Heraklion, Greece; 2 University of Bourgogne, Physics Laboratory, Dijon, France; 3 University of Montpellier, Institut National de la Santé et de la Recherche Médicale U414, France.

PURPOSE. To evaluate the feasibility of imaging normal corneal epithelium by means of atomic force microscopy (AFM).

METHODS. Twelve normal corneas from six albino rabbits were examined using a commercial atomic force microscope. Six corneas were examined in balanced salt solution after fixation in glutaraldehyde 2.5% and six without any fixation. Rectangular silicon nitride cantilevers with a spring constant of 10 to 20 mN/m were used. The measured forces after imaging were less than 100 pN. All reported images were made with 512 x 512-pixel definition with typical scan rates ranging from 1 to 5 Hz.

RESULTS. High-quality images of corneal epithelium surface were obtained from fixed and unfixed specimens in magnifications ranging from x2000 to x2,000,000. Imaging of fixed specimens was always easier. In unfixed specimens fuzzy images were very common, probably because of the presence of the cell glycocalyx. AFM revealed the typical polygonal corneal epithelial cells. The cell surface was covered by microprojections; at cell borders the microprojections were arranged in two characteristic parallel rows. Craterlike formations were revealed in several specimens. The microprojections’ morphology and their surface details were revealed using magnifications up to x2,000,000. Three-dimensional representation of the images facilitated better understanding of the surface topography. Measurements in horizontal and vertical plane were made using the section analysis tool.

CONCLUSIONS. In this work the AFM parameters appropriate for corneal epithelium imaging in physiological medium were defined. AFM represents a new powerful tool for corneal epithelium imaging, and its application in this field warrants further investigation.






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