Clara charge coupled device camera

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.

Immediately prior to imaging, yeast nitrogen base, casamino acids, uracil, tryptophan, and adenine (YPD) cultures were washed with YC complete + 2% filter sterile glucose and resuspended with YC complete + 2% filter sterile glucose. Yeast treated with benomyl were washed and resuspended with the YC complete + 2% filter sterile glucose with the same concentration of benomyl contained in the growth culture. Yeast were placed directly on glass coverslips and on glass slides. Coverslips were sealed with VALAP (equal parts Vaseline, lanolin, and paraffin). Imaging was performed at room temperature (25°C) using an Eclipse Ti wide-field inverted microscope (Nikon) with a 100 × Apo TIRF 1.49 NA objective (Nikon) and Clara charge-coupled device camera (Andor) using Nikon NIS Elements imaging software (Nikon). Images were acquired using the Scan Large Image program. Forty-nine z-stacks of seven z-steps with step size 300 nm were taken. At each interval a seven-step z-stack of 200-nm step size was acquired in the GFP (600 ms exposure), RFP (600 ms exposure), and Trans (25 ms exposure) channels. GFP, RFP, and Trans images were acquired before shifting Z position. All images were acquired from cells in the metaphase stage of the cell cycle, defined by the presence of a 1.2–1.7 µm spindle.