Scanr automated inverted fluorescent microscope system

Yeast strains were grown on synthetic media without uracil for selection. Imaging was performed using ScanR automated inverted fluorescent microscope system (Olympus). Images of cells were obtained in 384-well plates at 24°C using a 60 × air lens (NA 0.9) with an ORCA-ER charge-coupled device camera (Hamamatsu). Images were acquired in the GFP channel (excitation filter 490/20 nm, emission filter 535/50 nm).

The collection was visualized using an automated microscopy setup as described previously (Breker et al., 2013 (link)). In short, cells were transferred from agar plates into 384-well polystyrene plates for growth in liquid medium using the RoToR arrayer robot. Liquid cultures were grown in a LiCONiC incubator, overnight at 30°C in SD medium lacking uracil to select for yeast containing the plasmid encoding Ire1–mCherry. A JANUS liquid handler (PerkinElmer) connected to the incubator was used to dilute the strains to an OD₆₀₀ of ∼0.2 in plates containing SD plus DTT. Plates were incubated at 30°C for 2 h. The cultures in the plates were then transferred by the liquid handler into glass-bottom 384-well microscope plates (Matrical Bioscience) coated with concanavalin A (Sigma-Aldrich). After 20 min, wells were washed twice with SD −riboflavin complete medium to remove non-adherent cells and to obtain a cell monolayer. The plates were then transferred to the ScanR automated inverted fluorescent microscope system (Olympus) using a robotic swap arm (Hamilton). Images of cells in the 384-well plates were recorded in SD −riboflavin complete medium at 24°C using a 60× air lens (NA 0.9) and with an ORCA-ER charge-coupled device camera (Hamamatsu). Images were acquired in the mCherry channel (excitation filter 572/35 nm, emission filter 632/60 nm).