Deltavision elite widefield deconvolution microscope

Strains were grown overnight in SD-Ura media (CSM-Ura (Sunrise Science), Yeast nitrogen base with ammonium sulfate, 2% (v/v) glucose) at 30 °C, followed by 20-fold dilution into fresh SC-Ura media. After growth for 4–5 h at 30 °C to OD600 ~0.5, cells applied to a thin SD-Ura agar pad on a standard microscopy slide and imaged live on a DeltaVision Elite Widefield Deconvolution Microscope (GE Healthcare) using a 100× oil immersion objective with an sCMOS camera (Teledyne Photometics). Images were processed and analyzed in ImageJ^{66 (link)}.

Infected RBCs were harvested and resuspended in PBS at 5% hematocrit. Coverslips were coated with lectin (from Phaseolus vulgaris; PHA-E, Sigma product L8629), washed, and infected RBCs were applied. Adhered cells were washed three times with PBS until a monolayer remained on the coverslip. The cells were then fixed for 20 min in 4% paraformaldehyde/0.008% glutaraldehyde in PBS, followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min. Rat anti-HA antibody (1:300) in 3% (w/v) BSA in PBS was applied for 1.5 h, washed three times with PBS, followed by incubation with anti-rat AlexaFluor 568 (1:600) for 1 h. The nuclear stain DAPI (4′, 6-diamidino-2-phenylindole) was added prior to mounting and sealing of the slide with nail polish. Slides were imaged on a GE DeltaVision Elite Widefield Deconvolution Microscope. Deconvolved images were processed using FIJI software (62 (link)). Images stacks are presented as maximum projections and have been adjusted by cropping, adding false color and changes to brightness/contrast.