For confocal imaging of virus samples and cells, we use a Nikon Eclipse Ti microscope body, equipped with a Yokogawa CSU-X spinning disk, Andor Zyla sCMOS camera, 60X and 100X APO TIRF objectives (1.49 NA), and a Spectral Applied Research ILE-3003 laser launch for illumination via laser lines at 405nm, 488nm, 561nm, and 640nm. Because the vast majority of viruses immobilized on coverslips lie in-plane in our samples with a thickness less than our depth of focus, we are able to image the entire particle with a single z-slice. For data from different conditions within replicates, we imaged samples in immediate succession to avoid any effect from drift in the optical system over time; similarly, we acquired data from replicates to be combined typically within ~1 month of each other. For TIRF imaging, we use the same microscope body, objectives, and laser lines, but an Andor iXon Ultra EMCCD camera in place of the sCMOS camera for the increased sensitivity necessary for calibrating counts of molecules on the surface of labeled viruses (see “Quantifying HA and NA abundance ”).
Csu x spinning disk
Manufactured by Oxford Instruments
Sourced in Japan
The CSU-X spinning disk is a modular confocal scanning unit that provides high-speed and high-sensitivity imaging for live-cell applications. It uses a spinning disk design to rapidly scan the sample, enabling fast image acquisition with reduced phototoxicity. The CSU-X is compatible with a wide range of microscope systems and can be integrated into various imaging setups.
Automatically generated - may contain errors
3 protocols using csu x spinning disk
1
Confocal Imaging of Virus Samples
2
Live-cell Spinning Disk Microscopy
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Single-channel imaging was carried on a spinning disk confocal microscope (Olympus IX83, Olympus UPlanSApo 100×/1.40 oil objective, Yokogawa CSU-X spinning disk, Andor iXon Ultra 897 EMCCD camera) equipped with a temperature control unit (INU Stage Top Incubator, Tokai Hit, Shizuoka-ken, Japan). All culture dishes were kept in the chamber at 37 °C for 10 min before images were acquired. This was done to ensure that all dishes were imaged at the same time after labeling. Qdot 565 was excited using a 488 nm laser and acquired with an emission filter (600/50). Videos were acquired at a rate of 20 frames/s.
3
Live-Cell FRAP Imaging of GFP Dynamics
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Live imaging of GFP Fluorescent Recovery After Photobleaching (FRAP) in HEK-293T cells was performed with a Nikon-Ti microscope (X40 oil objective) paired with the Andor Frappa device, Yokogawa CSU-X spinning disk, and an iXon897 EMCCD camera. FRAP on complete cells was done using a 488nm laser at 15 mW power. Dual color images of GFP and Cy3 miRNAs were acquired prior to FRAP, and for 20 consecutive times afterward at 30-s intervals. Live imaging was performed in a controlled CO2 and 37 °C chamber. Image analysis was performed with FIJI.
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