Visualizing Yeast Spindle Dynamics

Yeast cells expressing Spc42-Cerulean were grown to log phase in SC medium supplemented with 2 mM of ascorbate, and imaged at 25 °C on a custom-built spinning-disk confocal microscope as previously described^{19 (link)}. Briefly, the following components were installed on a Leica DM6000 inverted microscope: a 100x/1.46 numerical aperture plan apochromatic objective, an XY stage with a Z top piezo (Applied Scientific Instrumentation), a Borealis head (a Quorum conversion of a Yokogawa QLC-100). Solid state lasers (446 and 515 nm) with an emission filter wheel were used for multicolor imaging. A Hamamatsu ImagEM EM-CCD camera was used for detection. MetaMorph (Molecular Devices) was used for image acquisition. Images were collected in a streaming regime as Z-stacks (200 or 300 nm Z-steps across 31 or 30 focal planes, respectively) with an integration time of 50 ms per focal plane. For spindle dynamics and alignment studies, cells were imaged over ten minutes with a ten second time step between stack acquisitions. For imaging astral MTs in live cells, strains containing Venus-Tub1 were imaged over five minutes with five second time step between stack acquisitions (the Spc42-Cerulean pole reporter was collected with ten second time steps in these strains).