Apo total internal reflection fluorescence 1.49 numerical aperture objective

Population and time-lapse images were acquired at room temperature (24°C) using a Nikon Eclipse Ti wide-field inverted microscope with a 100× Apo total internal reflection fluorescence 1.49 numerical aperture objective (Nikon, Melville, NY) and Andor Clara charge-coupled device camera (Andor, South Windsor, CT). Time-lapse stacks, each of seven 200-nm z-planes, were acquired every 30 s over a 10-min period (147 total planes per time lapse) with Nikon NIS Elements imaging software. Images were taken in transilluminated light, GFP and RFP fluorescence illumination. We chose the 30-s intervals as the best compromise between rate of acquisition and duration of time lapse due to photobleaching. The 30-s intervals were used in Verdaasdonk et al. (2013 (link)), where we report the effect of tethering on chromosome motion. Of importance, the MSD slopes of a 10-kb lacO/LacI-GFP array, 6.8 from CEN XV (KBY8065), taken at 3- and 30-s intervals are overlapping (Supplemental Figure S4). The 3-s interval allows one to estimate the slope of the MSD on a log-log plot, and the 30-s interval over a longer time scale provides a better estimate of the plateau value, which provides a better measurement of R_c.

Yeast cells containing the SPB protein Spc42-mCherry and the outer kinetochore protein Ndc80-GFP were imaged at 25°C on a Nikon Eclipse Ti wide-field inverted microscope with a 100×Apo total internal reflection fluorescence 1.49 numerical aperture objective (Nikon, Melville, NY) and Andor Clara charge-coupled device camera (Andor, South Windsor, CT). Images were acquired using Nikon Elements imaging software. Images were taken in transilluminated light and GFP and RFP fluorescence illumination. Metaphase spindles are in the length range of 1.4–1.7 µm.