Wide-field fluorescence microscopy imaging and analysis

Images of cells coexpressing Spc72-GFP and Spc42-CFP (as reference) were acquired with a Nikon Eclipse E800 with a CFI Plan Apochromat 100x, NA 1.4 objective, a Chroma Technology CFP/YFP filter set and a Coolsnap-HQ CCD camera (Roper Scientific) (Guo and Segal, 2017) as five-plane Z-stacks of fluorescence images at a distance of 0.8 μm between planes with 2 × 2 binning, paired to a DIC image at the middle focal plane (Juanes et al., 2013). Stacks were processed into 2-color overlays of maximal intensity 2-D projections and analyzed with MetaMorph software (Molecular Devices). Linescans for fluorescence intensity along the spindle axis were generated with the line tool set to 3-pixel width and normalized intensity plots produced in Microsoft Excel. Integrated intensities were determined in a 7 × 7 pixel region and cell background subtracted for each channel. An asymmetry index was calculated as the absolute difference between the relative values at each SPB (i.e. Spc72/Spc42 intensity ratio) divided by the sum of the same values. The index ranged from 0 (absolute symmetry) to 1 (absolute asymmetry). Statistical analysis was carried out using GraphPad Prism and the open source package R. Boxplots depict 5th, 25th, 50th, 75th and 95th centiles with notches indicating the 95% confidence interval of the median.