Alternative approaches to compensation of chromatic aberration lead to different Delta values in taxol-treated cells. (A) DIC images (top) and maximum intensity projections (bottom) of control and taxol-treated RPE1 cells in metaphase. Inner and outer layers of kinetochores are labeled using CenpA-GFP expression (green) and immunostaining for Hec1 (FluoroNanoGold, red). Spindle poles (centrioles) are labeled using expression of Centrin1-GFP (blue arrows). (B) Diagrams illustrating two alternative methods for calculating Delta measurements developed by Wan et al. (2009). Method I: pairwise calculations of sister kinetochores based on projecting inner kinetochore spots onto the axis determined by the outer kinetochore spots; method II: direct measurement of Delta after global chromatic correction based on the assumption that the spindle is symmetric overall. (C) Box plots presenting the values of Delta calculated by method I versus method II. ***, P < 0.001 in two-tailed t test. (D) Mean values ± SD for various parameters of kinetochore organization calculated by method II.