Abstract
Objective: The atomic force microscope (AFM) is a useful tool for studying proteins at the single molecule level, however, these studies in physiologically relevant conditions have been restricted, in part, by both the noise and speed limitations of the AFM. Recent progress in AFM with small cantilever enables us to observe individual protein interactions in faster and quieter conditions. Here we have observed, in real time, submembranous structures of the cytoplasmic side of plasma membrane of human umbilical vein cord endothelial cells (HUVECs). Methods: HUVECs cultured on quartz specimen holders were sonicated in hypotonic solution, then attached on the scanner of a high-speed AFM (NVB500, Olympus). The cantilevers used were typically 10 μm long, 2 μm wide, and 100 nm thick. Each cantilever had an electron beam deposited tip that was 600-800 nm long. All imaging was done in tapping mode to minimize sample damage and performed at ambient temperature. Results and Discussion: The cytosplasmic face of the fixed basal HUVEC plasma membrane showed meshwork structure and particles of various size (10–40 nm) at lower magnification (800 x 600 nm). Single actin filaments formed three-dimensional lattice at higher magnification (200 x 150 nm). These observations are consistent with our previous observation by confocal laser microscope and quick-freeze deep-etch electron microscope. The high-speed AFM will open the possibility of studying membrane proteins such as ion channels at single molecule level. [J Physiol Sci. 2007;57 Suppl:S119]
