ENDOTHELIAL-CELL ADHESION IN REAL-TIME - MEASUREMENTS INVITRO BY TANDEM SCANNING CONFOCAL IMAGE-ANALYSIS

Real time measurements of cell-substratum adhesion in endothelial cell s were obtained by tandem scanning confocal microscopy of sites of foc al contact (focal adhesions) at the abluminal cell surface. Focal cont act sites were sharply defined (low radiance levels) in the living cel l such that the images could be enhanced, digitized, and isolated from other cellular detail. Sites of focal contact are the principal deter minant of cell-substratum adhesion. Measurements of (a) the focal cont act area and (b) the closeness of contact (inverse radiance) were used to nominally define the adhesion of a single cell or field of cells, and to record spontaneous and induced changes of cell adhesion in real time. The topography of focal contacts was estimated by calculating s eparation distances from radiance values using a calibration technique based on interference ring optics. While slightly closer contact was noted between the cell membrane and substratum at or near the center o f each focal contact, separation distances throughout the adhesion reg ions were always < 50 nm. Subtraction of consecutive images revealed c ontinuous spontaneous remodeling of individual focal adhesions in unpe rturbed cells during periods of < 1 min. Despite extensive remodeling of focal contact sites, however, cell adhesion calculated for an entir e cell over extended periods varied by < 10%. When cytoskeletal stabil ity was impaired by exposure to cytochalasin or when cells were expose d to proteolytic enzyme, endothelial adhesion declined rapidly. Such c hanges were recorded at the level of single cells, groups of cells, an d at single focal adhesions. In both unperturbed and manipulated cells , the dynamics of remodeling and cell adhesion characteristics varied greatly between individual sites within the same cell; disappearance o f existing sites and appearance of new ones often occurred within minu tes while adjacent sites underwent minimal remodelling. Tandem scannin g confocal microscopy image analysis of living cells in real time prov ides repetitive spatial, temporal, and quantitative information about cell adhesion. Such an approach should allow more precise quantitative analyses to be made of the interactions between extracellular matrix, adhesion proteins, integrins, and the cytoskeleton in the living cell .