(a) Axially varying excitation intensity arises from interference between direct and reflected light off a silicon substrate. The structure of the illumination pattern is controlled by varying the polarization and angle of incidence of the excitation light, and is manipulated to probe the height of a fluorophore above a reflective substrate. A thin layer of oxide is used to space the sample above the silicon surface. (b) Average emission intensity and fits to the optical model for beads at the indicated axial positions, and (c) the corresponding histograms of axial position measured for individual beads. (d) Measured axial position of fluorescent beads adsorbed on silicon oxide layers of varying thickness plotted against their expected position. Data are plotted as the mean, horizontal bars show standard deviation σ (n = 45 - 50). Height is reported as the axial distance of the bead center above the reference point z = 500 nm. (e) Epifluorescence image (Membrane) and three-dimensional scanning angle interference reconstruction (Topography and Zoom) of an epithelial cell ventral membrane labeled with DiO. Boxes in the epifluorescence image indicate regions shown in ROI (larger box) and Zoom (smaller box). Height is reported as the absolute distance of the membrane above the silicon oxide surface. Scale bars, 2 μm (ROI), 750 nm (Zoom).

(a) Epifluorescence image (left panel) of microtubules in a fixed epithelial cell. The red box marks the region of interest (ROI) shown in the right panels; immunofluorescence (top) and the corresponding scanning angle interference image (bottom) are shown. (b) The schematic shows dual-labeled talin, which binds to integrin and actin through its FERM and THATCH domains, respectively. The images show the difference in height of the integrin-binding N-terminus of talin and the actin-binding C-terminus. Scale bars – 5 μm.

(a, b) Live-cell time-lapse multicolor scanning angle interference imaging of epithelial cells expressing paxillin-mEmerald and mCherry-vinculin following cell-cell contact (a) or in an assembling adhesion during cell retraction (b). The left panels show paxillin epifluorescence images at the indicated time point, the region of interest (ROI) is boxed in red. The middle and right panels show heights of paxillin and vinculin in the ROI. The plots show distributions of paxillin and vinculin heights in ROI, mean height (H) above the oxide surface is calculated over 35 to 120nm. In (a) the histogram shows the measured heights of paxillin and vinculin at each pixel corresponding to site of adhesion. In (b) the histogram displays the heights of the molecules at the subset of ROI pixels within the boundary of the left adhesion (indicated by white arrow). (c) The plot shows the relative heights of paxillin and vinculin in maturing adhesions during cell retraction. Axial position is reported relative to the average height of molecules in all adhesions in the cell (P < 0.001; n=3 cells, >= 9 adhesions).