All micrographs in the main text were acquired on a CSU-X spinning-disk confocal microscopy system (Yokogawa, Intelligent Imaging Innovations) with a DMI6000B microscope (Leica), 100 × 1.45 NA objective (Leica), and an Evolve 512Delta EMCCD (Photometrics) with a 2× magnifying lens (Yokogawa) for a final resolution of 0.084 µm/pixel. All images and videos shown are transverse, single-plane cross-sections, unless stated otherwise. For live-cell imaging of yeast, cells in mid–log phase were adhered to a glass-bottomed dish (CellVis) coated with concanavalin A (EY laboratories) and washed with the respective cell medium. Molecule counting for Rgd3 was performed by comparison to Cse4 puncta intensity at anaphase, as described previously (Donovan and Bretscher, 2012 (link)). Imaging at elevated temperatures was performed in an environmental chamber (Okolab) following 1-h incubations in a 37°C water bath, except for the tpm1-2 tpm2∆ experiment, which was performed in a CherryTemp chamber (Cherry Biotech) for rapid temperature shift. Images were analyzed and processed with Slidebook 6.0 software (Intelligent Imaging Innovations) or FIJI. Images and figures were assembled in Illustrator (Adobe).
1.45 na objective
Manufactured by Leica
The 100 × 1.45 NA objective is a high-magnification, high-numerical aperture objective lens designed for Leica microscopes. This objective provides a magnification of 100× and a numerical aperture of 1.45, which are key specifications that determine the resolving power and light-gathering ability of the lens.
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3 protocols using 1.45 na objective
1
Yeast Cell Imaging Protocols
2
Live and Fixed Cell Imaging of Yeast
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For live-cell imaging, yeast were grown in liquid culture to mid-log phase (0.5–0.8 OD) and were prepared by adhering 200 µl of cells to 4-well glass-bottom dishes (CellVis) coated with concanavalin-A submerged with proper media for cell survival. For fixed imaging, yeast were grown in liquid culture to mid-log phase and 4% paraformaldehyde was added to the culture for 10 min. Cells then followed appropriate protocol (see Actin staining) and then were adhered to coverslips, again by using concanavalin-A. Coverslips were then mounted using soft mounting media (Invitrogen) and secured by using clear nail polish. Some fixed cells were also imaged using agarose pads, where 4 µl of fixed cells were added to a 1% agarose pad and media was allowed to diffuse into the agarose (Pringle et al. 1989 (link); Shin et al. 2018 (link)). All fluorescent microscopy was conducted on an inverted microscope (Leica DMI6000B) which had a spinning disk confocal unit (Yokogawa CSU-X1) with 100× 1.45 NA objective (Leica) with either a Evolve 512Delta EMCCD or with a Flash 4.0v2 CMOS camera. DIC imaging was conducted on this microscope as well as on a Leica De-Convolution Microscope (DMi8). Slidebook 6.0 or FIJI software were used for image analysis.
3
Fluorescence Recovery After Photobleaching
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4XUb-Cy5 and Hse1-Oregon Green fluorescence recovery after photobleaching (FRAP) were performed on a CSU-X spinning-disk confocal microscopy system (Intelligent Imaging Innovations), coupled to a DMI 6000B microscope (Leica), 100×/1.45-NA objective, and a QuantEMCCD camera. Analysis was performed on Slidebook and ImageJ. Three whole droplets were bleached with either the 647-nm or the 488-nm laser. Fluorescence values of unbleached droplets were taken to correct for photobleaching while imaging.
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