Figure 1. Scanning Angle Interference Microscopy.

(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).