Shapes of CenpT and Hec1 distributions visualized via SI microscopy. (A) Direct comparison of conventional WF versus SI microscopy. In WF, microscopy (top) of sister kinetochores with lower (a′) and higher (a″) Delta appear as individual round spots of CenpT and Hec1 that are shifted with by the value of Delta with respect to one another. SI (bottom) reveals complex multilobe distributions of CenpT and Hec1 within the diffraction limited spots. Full optical Z series for each kinetochore are shown in both WF and SI. Distance for each plane from the central slice is indicated. Illum, illumination. Bars: (left) 1 µm; (right) 200 nm. (B and C) SI images of kinetochores (maximum intensity projections) representative of the low (left panels) versus high range of Delta values (right panels) in control (B) and taxol-treated (C) metaphase cells. The images are aligned so that the outer layer always faces to the right and centroids of CenpT (red dots) and Hec1 (green dots) separate along the horizontal axis. Red arrowheads denote lobes of CenpT that extend beyond Hec1 toward microtubules. Green arrowheads point at protuberances in the distribution of Hec1. Measured values of Delta (method II) are shown above the panels. Bars, 200 nm.