Integration of total internal reflection microscopy and atomic force microscopy (TIRFM-AFM) to study mechanotransduction in endothelial cells

Atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) were used simultaneously to determine the effect of localized force application on cell-substrate contact size and position. Fluorescently labeled Human Umbilical Vein Endothelial Cells (HUVECs) were cultured on glass converslips for 24 h and the basal and apical surfaces were mapped using TIRFM/AFM. Several AFM-TIRFM experiments were conducted at an interfacial angle of 72°. Silicon nitride cantilevers with spring contacts were used for contact mode AFM imaging under fluid. Forces ranging from pico-newtons to nano-newtons were applied to the cell surface near the nucleus and away from the nucleus. Results of the force application study and the force volume measurements show that the novel TIRFM-AFM technique provides a unique method to study the mechanisms of stress transduction for anchorage-dependent cells.