For all microscopy experiments, cells were grown to mid-log, washed, and resuspended in SD-N for the time points indicated. Except for Figure 2B , deconvolved images were obtained using a Nikon microscope (Model E800) with a 100× objective using 1.2× camera magnification (Plan Fluor Oil, NA 1.3) and a CCD camera (Hamamatsu Model C4742). Data were collected using NIS software and processed using Image Pro software. All images of individual cells were optically sectioned (0.2-μm slices at 0.3 μm spacing) and deconvolved, and slices were collapsed to visualize the entire fluorescent signal within the cell. Linear quantification analysis was measured using the Image Pro software. The vacuoles were visualized in live cells either using mCherry-tagged Vph1, Pho8-BFP or staining with CMAC (Thermo, C2110; 100 μM) as described [21 (link)]. In Figure 2B a Keyence BZ-X710 fluorescence microscope with a 100× objective with 1.0× camera magnification (PlanApoλ Oil, NA 1.45) and a CCD camera was used. The images were deconvolved using BZ-X Analyzer software.
Model e800
Manufactured by Hamamatsu Photonics
The Model E800 is a spectrophotometer designed for measuring the optical absorption and transmission properties of various materials and samples. It is capable of operating in the ultraviolet, visible, and near-infrared wavelength ranges. The instrument features a monochromator for wavelength selection and a photodetector to measure the light intensity.
Automatically generated - may contain errors
2 protocols using model e800
1
Microscopy Analysis of Cellular Organelles
2
Quantitative Live-cell Microscopy Protocol
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For all microscopy studies, cells were grown to mid-log phase, washed, and resuspended in SD-N with rapamycin for the time points indicated. Deconvolved images were obtained using a Nikon microscope (Model E800) with a ×100 objective with ×1.2 camera magnification (Plan Fluor Oil, NA 1.3) and a CCD camera (Hamamatsu Model C4742). Data were collected using NIS software and processed using Image Pro software. All images of individual cells were optically sectioned (0.3-μM spacing), deconvolved and slices were collapsed to visualize the entire fluorescent signal within the cell. The nuclei were visualized in live cells using either the nuclear marker Nab2-mCherry or the nuclear pore marker Nup1-mCherry. Linear quantification analysis was measured using the NIS software. Single plane images were obtained using a Keyence BZ-X710 fluorescence microscope with a ×100 objective with ×1.0 camera magnification (PlanApoλ Oil, NA 1.45) and a CCD camera. Data were collected using BZ-X Analyzer software.
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