Na plan apochromat oil immersion objective

Spinning-disk confocal microscopy (SDCM) was performed on one of two systems. SDCM #1 was a Nikon Eclipse Ti inverted scope equipped with PerkinElmer UltraVIEW VOX confocal system (Perkin Elmer) and an EM-CCD camera (Hamamatsu) housed within a 37°C environment chamber. Cells were excited with 405nm, 488nm, 561nm, and 640nm laser lines, and emission was collected using either a 100X/1.49NA apochromat TIRF objective (Nikon) or 60X/1.4NA plan apochromat oil immersion objective (Nikon) with standard emission filter sets. Images were acquired with Volocity acquisition software (Perkin Elmer). Z-stacks were acquired using a piezo motor and step sizes of either 100 nm or 150 nm, requiring ~200–300 steps to fully sample rounded, metaphase cells. 3D image stacks were deconvolved with Huygens Professional v. 19.04 (Scientific Volume Imaging) with the CMLE algorithm and signal-to-noise ratio between 20 and 30, maximum of 40 iterations, and stopping criterion of 0.01. SDCM #2 was a NikonTiE inverted microscope equipped with a spinning-disk scan head (Yokogawa, CSU-X1) and an EM-CCD camera (iXon Ultra 897). Imaging was performed with standard laser-lines with fluorescence emission collected by a 100X/1.49NA apochromat TIRF objective (Nikon). Images were acquired with NIS-Elements AR v4.40.00 (Nikon). Cells were incubated within a Tokai Hit stage top incubator at 37 °C and 5% CO₂.