Sutter dg4 light source

For all microscopy experiments, overnight cultures grown at 30 C in YPD medium were re-suspended at a final OD₆₀₀ of 0.2 and allowed to reach mid-log phase prior treatment and image acquisition. Live cell imaging was performed by briefly centrifuging the cells (800 g for 3 min), followed by resuspending in a minimal volume of growth media, spotting onto glass slides and coverslipping prior to imaging. All images were acquired using a Leica DMI 6000 florescent microscope (Leica Microsystems GmbH, Wetzler Germany), equipped with a Sutter DG4 light source (Sutter Instruments, California, USA), Ludl emission filter wheel with Chroma band pass emission filters (Ludl Electronic Products Ltd., NY, USA) and Hamamatsu Orca AG camera (Hamamatsu Photonics, Herrsching am Ammersee, Germany). Images were acquired at 0.2 µM steps using a 63× oil-immersion objective with a 1.4 numerical aperture. Deconvolution and analysis were performed using Velocity Software V4 (Perkin Elmer). For most images, representative images of the middle section and compressed image stack are shown. Numerical insets represent the indicated quantifications of at least 100 cells from 2 to 3 independent experiments unless indicated otherwise.

Cells from mid-logarithmic cultures were collected, washed, and fixed in ice cold 70% ethanol. The cells were recollected and resuspended in 1X PBS containing 1 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. The cells were pelleted then resuspended in 1X PBS and spotted on microscopy slides. Stained cells were viewed under 100× magnification using the Leica DMI 6000 florescent microscope (Leica Microsystems, Germany), equipped with a Sutter DG4 light source (Sutter Instruments, CA, USA), Ludl emission filter wheel with Chroma band-pass emission filters (Ludl Electronic Products, NY, USA) and Hamamatsu Orca AG camera (Hamamatsu Photonics, Japan). Multiple images were captured using Volocity 4.3.2 Build 23 (Perkin-Elmer, MA, USA).

BY4743 cells were grown overnight at 30° in YPD. Cultures were then reinoculated into 50 mL of YPD to an OD₆₀₀ of 0.1 and incubated at 30°. Once they reached log phase, cultures were washed twice in YP and resuspended in 10 mL of YP. Cell suspensions were diluted to an OD₆₀₀ of 0.1 in 50 mL of indicated media and incubated for 6 days at 30°. On each day, 100 μL was aliquoted from each culture for cell counting on a hemocytometer. In addition, 5 mL was aliquoted from each culture, spun down, and resuspended in SC (no dextrose) for brightfield imaging (0.006 s exposure time, 100% gain) using the Leica DMI 6000 fluorescent microscope (Leica Microsystems GmbH, Wetzler, Germany) equipped with a Sutter DG4 light source (Sutter Instruments, CA, USA), Ludl emission filter wheel with Chroma band pass emission filters (Ludl Electronic Products Ltd., NY, USA) and the Hamamatsu Orca AG camera (Hamamatsu Photonics, Herrsching am Ammersee, Germany).