Plan apo oil objective

Imaging was performed with a Nikon Eclipse Ti inverted microscope equipped with an Orca R2 (Hamamatsu) camera, a 60X Plan Apo oil objective (NA 1.4, Nikon), an automated stage (Ludl), a Lumencor SOLA fluorescent illumination system, and a custom-designed temperature-controlled Plexiglas enclosure in which the temperature was maintained at approximately 37°C during imaging. Image acquisition was performed using MATLAB scripts interfacing with μManager, as previously described [29 (link)]. Semrock filter cubes for GFP (GFP1828A) and mKate2 (mCherry-B) were used to image mNeonGreen and mNeptune, respectively. mNeonGreen (used for σ^B reporters) was imaged with 2x2 pixel binning at approximately 20% illumination power with 200-ms exposures, and mNeptune (used for cell segmentation) was imaged with 1x1 binning at approximately 24% power with 400-ms exposures). Images were captured at 10-minute intervals.

Confocal images were acquired on a Nikon (Melville, NY) A1 confocal microscope, with a 100× Plan Apo oil objective. NIS Elements imaging software was used to control image acquisition. Images were further processed using the Fiji distribution of ImageJ (Schindelin et al., 2012 (link)). GraphPad Prism was used for statistical analyses.
Vacuolar localization of yEGFP–Pib2 constructs was quantified using Fiji. For each cell, vacuolar membrane and cytosolic regions of interest (ROIs) of equal area were determined and the fluorescence within those ROIs was measured. FM4-64 fluorescence was used to determine the location of the vacuolar membrane ROIs. Within each image, an average background fluorescence was determined and subtracted from the vacuolar and cytosolic intensity measurements. The localization was then expressed as a ratio of vacuolar membrane fluorescence to cytosolic fluorescence. For statistical analyses, a ROUT outliers test (Q=0.1%) was used and data were further assessed by one-way ANOVA with Tukey multiple comparisons.
To determine expression levels of targeting constructs, cells were imaged in widefield using a Nikon Ti Microscope with an S Plan Fluor ELWD 20× objective. NIS Elements imaging software was used to control image acquisition. Images were processed in NIS Elements imaging software using a custom macro. GraphPad Prism was used for statistical analyses.

Imaging was performed with a Nikon Eclipse Ti inverted microscope equipped with a Photometrics Prime 95B sCMOS camera, a 100× Plan Apo oil objective (NA 1.45, Nikon), an automated stage (Nikon), a Lumencor SOLA SE II 365 Light Engine fluorescent illumination system, and an OKO temperature-controlled enclosure in which the temperature was maintained at approximately 37°C during imaging. Image acquisition was performed using NIS-Elements AR 5.11.03 64-bit. Filter cubes for GFP and mCherry were used to image mNeonGreen and mNeptune, respectively. mNeonGreen (used for σ^B reporters) was imaged at approximately 33% illumination power with 200-ms exposures, and mNeptune (used for cell visualization) was imaged with at approximately 33% power with 400-ms exposures. The images were captured at 10-min intervals. Phase-contrast images were occasionally also captured.

Light microscopy was performed on a Nikon Eclipse Ti inverted microscope equipped with a Photometrics Prime 95B sCMOS camera, a 100× Plan Apo oil objective (NA 1.45, Nikon), an automated stage (Nikon), a Lumencor SOLA SE II 365 Light Engine fluorescent illumination system, and an OKO temperature-controlled enclosure in which the temperature was maintained at approximately 37°C during imaging. Image acquisition was performed using NIS-Elements AR 5.11.03 64-bit. mNeonGreen (used for σ^B reporters) was imaged at approximately 33% illumination power with 200 ms exposures, and phase contrast was used for cell visualization. Images were captured at 10-min intervals. Cell lineages were manually curated, tracked, and plotted as in our previous work (22 (link)).