Csu x high speed confocal scanner unit

All live-cell and fixed-cell confocal imaging was carried out on a Nikon Ti-E inverted microscope equipped with a Yokogawa CSU-X high-speed confocal scanner unit and a pair of Andor iXon 512 × 512 EMCCD cameras. High-magnification images were obtained using a 100× 1.49 NA oil-immersion objective, while lower magnification images for high-content imaging were acquired through a 40× 1.3 NA oil immersion objective. Images were typically acquired with 50× EM gain and 100-ms exposure. The 405-nm laser was operated at 10 mW, while 488-mW and 561 lasers were operated at 25 mW. All components of the microscope were controlled by the µManager open source platform (52 ). The microscope stage was enclosed in a custom-built incubator that maintained preset temperature and CO₂ levels for prolonged live-imaging experiments. To avoid unintentional selection bias (such as picking cells with higher IRE1 expression levels or more visible IRE1 clusters), fields-of-view were selected by only looking at stained cell nuclei in the 405-nm channel. No cells or fields of view were subsequently excluded from analysis, ensuring that the data faithfully capture the distribution of IRE1 fluorescence across the entire cell population.

Confocal imaging was carried out on a Nikon Ti-E inverted microscope equipped with a Yokogawa CSU-X high-speed confocal scanner unit and an Andor iXon 512×512 EMCCD camera. All images were acquired through a 40×1.3 NA oil immersion objective. Images were typically acquired with 60×EM gain and 100 ms exposure. The four lasers used were 405 nm (operated at 10 mW), 488 nm (operated at 25 mW), 561 nm (operated at 25 mW), and 640 nm (operated at 15 mW). All components of the microscope were controlled by the μManager open-source platform (Edelstein et al., 2010 (link)). The microscope stage was enclosed in a custom-built incubator that maintained preset temperature and CO₂ levels for prolonged live-imaging experiments. To avoid unintentional selection bias, fields-of-view were selected by only looking at stained cell nuclei in the 405 nm channel. No cells or fields-of-view were subsequently excluded from analysis, ensuring that the data faithfully capture the distribution of fluorescence across the entire cell population.