Apo tirf 60 oil objective

A single colony from a YPD plate containing yTK32 was inoculated into SC-Glu media and grown overnight in 24 well plates. The overnight culture was inoculated into 1 mL SC-Glu in 1:500, 1:1000, 1:2000 dilutions. These cultures were set to grow overnight at 30°C in 24 well plates at 200 RPM. Once the cells reached mid-exponential phase (OD₆₀₀ = 4.5), cells were diluted 1:50 in SC-Glu in a fresh 24-well plate. Cells were then adhered to 96-well plates pretreated with concanavalin A. One hour prior to imaging, adhered cells were washed three times with 150 μL GE media, leaving at least 50 μL of media in the well each time to avoid cell stress. Yeast were then imaged in 200 μL of GE media.
Live yeast timelapse images were acquired on the Nikon AX NSPARC using an Apo TIRF 60× Oil objective (NA = 1.49) with a 5× magnification for a pixel size of 0.057 μm. Yeast cells were imaged in 3D (40–70 z sections, 0.17 μm apart). The GFPmut3 and mCherry fluorophores were excited with 488 nm and 561 nm illumination respectively.
Yeast timelapse images were deconvolved using Nikon Elements and then processed in MitoGraph to obtain 3D renderings. For each single cell, the ROI was copied across channels and timepoints in order to keep the resulting renderings aligned. The cell surface was estimated using a Delaunay 3D filter on the rendering of the ER channel.