Samples were prepared as previously described35 (link). Image acquisition was performed on a Nikon TE300 inverted microscope equipped with a Yokogawa CSU10 spinning disk confocal with image magnification yielding a 65 nm pixel size from the Orca ER cooled CCD camera and an 100X/1.4NA (Plan Apo) DIC oil immersion objective (Nikon). Sixty-five flame 3D stacks of pairs of red and green fluorescent images were obtained sequentially at 200 nm steps along the z-axis through the cell from coverslip surface using MetaMorph 6.1 software (Molecular Devices).
For each metaphase kinetochore pair, 3D centroid positions were measured with a 3D Gaussian fitting function as described previously36 (link). The centroids of one color were projected to the axis defined by the centroids of the other color, and the Delta (average separation of the projection distance between the signals of red and green colors for theta pair) was calculated to correct for chromatic aberration. Mean Delta values were corrected for tilt of the face of the kinetochore relative to the axis between sister kinetochores.
For each metaphase kinetochore pair, 3D centroid positions were measured with a 3D Gaussian fitting function as described previously36 (link). The centroids of one color were projected to the axis defined by the centroids of the other color, and the Delta (average separation of the projection distance between the signals of red and green colors for theta pair) was calculated to correct for chromatic aberration. Mean Delta values were corrected for tilt of the face of the kinetochore relative to the axis between sister kinetochores.
