Imaging of cell/substrate contacts on polymers by total internal reflection fluorescence microscopy.

A simplified model of total internal reflection fluorescence (TIRF) emission of fluorescently labeled cell membranes [Reichert, W. M.; Truskey, G. A. J. Cell Sci. 1990, 96, 219-230] was used to determine the topography of the cell membrane in apposition to a polymer-coated surface. The homopolymer substrates were spun cast films of hydrophilic poly(hydroxyethyl methacrylate) (polyHEMA) or hydrophobic poly(ethyl methacrylate) (polyEMA). Bovine aortic endothelial cells (BAEC) on preadsorbed fibronectin polymer substrates were either plated for 24 h, fixed, labeled, and examined by TIRF microscopy (TIRFM) and phase-contrast microscopy or plated for 2 h and tested for their adhesion strength in a parallel-plate flow chamber. BAEC attached to polyHEMA showed no evidence of focal contact formation. However, BAEC attached to polyEMA were well spread and showed an array of focal contacts. TIRFM data were transformed to construct a detailed topographical map of relative cell/substrate separation distances. Virtually all of the BAEC plated to polyHEMA were sheared from the surface when subjected to a 50 dyn/cm2 burst of laminar flow, whereas only 10% of the BAEC were sheared from the polyEMA surface. These data suggest that the polyHEMA and polyEMA surface properties (e.g., hydrophobicity) correlate with the presence of BAEC focal contacts and the BAEC attachment strength.