Cells were incubated at 30°C in synthetic medium and grown to mid log phase. Cells were visualized using an Olympus IX71 microscope (Olympus) equipped with a CSU10 spinning-disk confocal scanner (Yokogawa Electric Corporation), as described previously (Yorimitsu and Sato, 2012 (link)). Images were captured using an electron-multiplying charge-coupled device camera (iXon, DV897; Andor Technology). Photoshop (Adobe) and ImageJ (National Institutes of Health) were used to prepare the images for figure preparation.
Csu10 spinning disk confocal scanner
Manufactured by Yokogawa
Sourced in Japan
The CSU10 spinning-disk confocal scanner is a compact and versatile laser scanning unit designed for fluorescence microscopy applications. The device utilizes a rotating microlens disk and a Nipkow disk to achieve high-speed confocal imaging with a wide field of view. The CSU10 is capable of capturing real-time confocal images at high frame rates, making it suitable for live-cell imaging and other dynamic sample analyses.
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3 protocols using csu10 spinning disk confocal scanner
1
Visualizing Cells Using Spinning Disk Confocal Microscopy
2
Fluorescence Microscopy of Yeast Cells
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Yeast cells expressing fluorescent protein-fused proteins were grown to mid-log phase at 30°C. Fluorescence microscopy observations were carried out using an Olympus IX71 microscope (Olympus, Tokyo, Japan) equipped with a CSU10 spinning-disk confocal scanner (Yokogawa Electric Corporation, Tokyo, Japan) and an electron-multiplying charge-coupled device camera (iXon, DV897; Andor Technology, South Windsor, CT, USA). The acquired images were analyzed by Andor iQ (Andor Technology, South Windsor, CT, USA). In this setting, a 473 nm solid-state laser (J050BS; Showa Optronics, Tokyo, Japan) was used to excite GFP and a 561 nm solid-state laser (J050YS; Showa Optronics, Tokyo, Japan) was used to excite mCherry.
3
Visualizing Kinetochore Dynamics in C. elegans
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Gravid adult hermaphrodite worms were dissected in M9 buffer, early embryos were transferred onto a 2% agarose pad using a mouth pipet, covered with a 22×22 mm coverslip, and imaged at 20°C. To assay kinetochore recruitment, embryos were imaged on an Andor Revolution confocal system (Andor) coupled to a CSU-10 spinning disk confocal scanner (Yokogawa) and an electron multiplication back-thinned charge-coupled device camera (iXon, Andor), using a 100× 1.4 NA Plan Apochromat objective; or with an inverted Axio Observer Z1 (Zeiss) microscope with a CSU-X1 spinning disk head (Yokogawa) and a 100× 1.3 NA Plan Apochromat Lens (Zeiss). A 6×2 μm z-stack was collected every 10 or 20 seconds.
For assaying mitotic timing and chromosome dynamics, one-cell embryos expressing GFP::H2b were imaged on a widefield deconvolution microscope (DeltaVision) connected to a charge-coupled device camera (pco.edge 5.5 sCMOS; PCO) and a 60× 1.42NA PlanApo N objective (Olympus). A 5×2 μm z-stack was collected every 10 seconds.
For assaying mitotic timing and chromosome dynamics, one-cell embryos expressing GFP::H2b were imaged on a widefield deconvolution microscope (DeltaVision) connected to a charge-coupled device camera (pco.edge 5.5 sCMOS; PCO) and a 60× 1.42NA PlanApo N objective (Olympus). A 5×2 μm z-stack was collected every 10 seconds.
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