Observation of human retinal pigment epithelium cell cultures using atomic force microscopy

Abstract

Abstract: : Purpose:To evaluate the feasibility of imaging human RPE cell surface in cell culture by means of atomic force microscopy(AFM). Methods:We used the immortalized human RPE cell line D407. Human RPE cells were examined using a commercial atomic force microscope. The RPE cells were examined in balanced salt solution either alive in their nutritional medium or 15 minutes after fixation in glutaraldeyde 2.5%. Rectangular silicon nutride cantilevers with a spring constant of 10 to 20 mN/m were used. The measured forces during imaging were less than 100 pN. All reported images were made with 512x512 pixel definition with typical scan rates ranging from 1 to 5 Hz. Results:Images of RPE cells were obtained from fixed and unfixed specimens in magnifications ranging from x2000 to 100,000. Imaging of fixed specimens was always easier and of better resolution. In unfixed specimens fuzzy images were very common, probably because of the presence of the cell glycocalyx. The cell shape and the other characteristics of RPE cells are comparable to those described in the literature using Scanning Electron Microscope. Apical microvilli as well as cell processes involved in cell–to–cell adhesion were depicted in several occasions and were studied in high resolution. Conclusions:AFM represents a new powerful tool for RPE cell imaging and its application in this field warrants further investigation. AFM images can reach very high magnification with a resolution comparable or better to SEM.

Keywords: microscopy: confocal/tunneling • retinal pigment epithelium • retina