All confocal images were acquired using a spinning disk confocal system (Andor Technology) consisting of a CSU-22 confocal (Yokagawa) and a Zyla 5.5 sCMOS camera (Andor Technology) mounted on an Olympus IX-81 inverted microscope with laser excitation (Coherent) and emission filters (Semrock). A 60 × objective (1.42 numerical aperture) oil-immersion objective with additional 1.6 × magnification in the light path and 1.2 × magnification placed between the confocal and the camera was used to yield a final 67-nm pixel size. Acquisition was controlled by iQ software (Andor). For live-cell experiments, cells were imaged in extracellular buffer (EB) containing (in mm ): NaCl, 120; KCl, 3; CaCl2, 2; MgCl2, 2; glucose, 10; HEPES, 10; pH adjusted to 7.35 with NaOH. Latrunculin A (LatA) was applied as a 40 × solution (200 µm , diluted in EB from a 20 mm stock solution in dimethylsulphoxide (DMSO) to the imaging bath resulting in a final concentration of 5 µm (and a final concentration of 0.025% DMSO).
Spinning disk confocal system
Manufactured by Oxford Instruments
Sourced in Japan
The Spinning disk confocal system is a laboratory instrument that enables high-speed, high-resolution imaging of biological samples. It utilizes a spinning disk containing multiple pinholes to illuminate and collect light from the sample, allowing for optical sectioning and improved image quality.
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Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Ixon3 emccd camera, by Oxford Instruments (2 mentions)
Csu w1, by Yokogawa (2 mentions)
Zyla 5.5 scmos camera, by Oxford Instruments (1 mentions)
Iq software, by Oxford Instruments (1 mentions)
Csu x1 spinning disk unit, by Yokogawa (1 mentions)
Zen 2, by Zeiss (1 mentions)
Te2000 e inverted microscope, by Nikon (1 mentions)
Emccd camera, by Nikon (1 mentions)
Scmos camera, by Oxford Instruments (1 mentions)
Apotome 2, by Zeiss (1 mentions)
Orca er camera, by Hamamatsu Photonics (1 mentions)
Te2000 e, by Nikon (1 mentions)
Zen blue, by Zeiss (1 mentions)
Imaris, by Oxford Instruments (1 mentions)
7 protocols using spinning disk confocal system
1
Spinning Disk Confocal Microscopy for Live-Cell Imaging
2
Live-Cell Imaging of Lysosome Dynamics
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For live‐cell imaging, 1 × 105 the indicated cells were plated and transfected in fibronectin coated 35 mm Mattek dishes. The following day, if required, lysosomes were labelled with 20 nM lysotracker‐Red and cells were imaged at a rate of 1 frame per second either using an inverted Nikon A1 confocal system with a 100× objective lens or an inverted CSU‐X1 Spinning Disk Confocal system with an Andor Ixon3 EM‐CCD camera and a 100× objective lens and when required a 1.6× auxiliary magnifier, both equipped with temperature and CO2 control and running NIS Elements. Movies were processed using NIS elements and Image J. Figures were assembled using Image J in conjunction with Adobe Photoshop and Illustrator packages (Adobe, CA, USA). Correlation analysis was performed by extracting individual frames, thresholding to remove background and applying the Image J Intensity Correlation Analysis plugin, comparing the first frame of the movie with sequential frames at the indicated time increments 65 .
3
Spinning Disk Confocal Imaging of GCaMP6m and Scarlett
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The light path used to image GCaMP6m and Scarlett at single cell resolution is an Andor spinning disk confocal system. Light supplied from a 150 mW 488 nm laser and a 50 mW 560 nm laser passes through a 5000 rpm Yokogawa CSU-X1 spinning disk unit with a Borealis upgrade (with a dual-camera configuration). A 40 x/1.15NA CFI Apo LWD Lambda water immersion objective (Nikon) with a P-726 PIFOC objective piezo (PI) was used to image the volume of the worm’s head. A custom quad dichroic mirror directed light emitted from the specimen to two separate Andor Zyla 4.2 USB3 cameras, which had in-line emission filters (525/50, and 625/90). Data was collected at 2 × 2 binning in a 512 × 512 region of interest in the center of the field of view.
4
High-Resolution Confocal Imaging of Skin
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Confocal images were acquired using a spinning disk confocal system (Andor Technology Ltd) equipped with an Andor Zyla 4.2 and a Yokogawa CSU-W1 (Yokogawa Electric, Tokyo) unit based on a Nikon TE2000-E inverted microscope. Four laser lines (405, 488, 561 and 625 nm) were used for near simultaneous excitation of DAPI, Alexa448, RRX and Alexa647 fluorophores. The system was driven by Andor IQ3 software. Tiled imaging was performed to sample 2 mm2 areas of skin. Stacks of 1 mm steps were collected with a 20x/0.75 CFI Plan-Apochromat air objective. Zen 2.3 software (blue edition, Carl Zeiss Microscopy GmbH, 2011) was used to stitch the acquired images. 40x oil objective was used to acquire z stacks of 0.5–1 mm steps.
5
Live-cell Imaging of Chlamydia Inclusions
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Fluorescence images were acquired using a Nikon spinning disk confocal system with a 60x oil-immersion objective, equipped with an Andor Ixon EMCCD camera, under the control of the Nikon elements software. Images were processed using the image analysis software ImageJ (http://rsb.info.nih.gov/ij/ ). Representative confocal micrographs displayed in the figures are maximal intensity projections of the 3D data sets, unless otherwise noted.
Live cell imaging of inclusions expressing the fluorescent protein Clover (Lam et al., 2012 (link)) under the control of the hctA promoter (Grieshaber et al., 2012 (link); Chiarelli et al., 2020 (link)) was achieved using an automated Nikon epifluorescent microscope equipped with an Okolab (http://www.oko-lab.com/live-cell-imaging ) temperature controlled stage and an Andor Zyla sCMOS camera (http://www.andor.com ). Images were taken every fifteen minutes for 48 hours. Multiple fields of view of multiple wells of a glass bottom 24 well plate were imaged. The fluorescence intensity of each inclusion over time was tracked using the ImageJ plugin Trakmate (Tinevez et al., 2016 (link)) and the results were averaged and plotted using python and matplotlib.
Live cell imaging of inclusions expressing the fluorescent protein Clover (Lam et al., 2012 (link)) under the control of the hctA promoter (Grieshaber et al., 2012 (link); Chiarelli et al., 2020 (link)) was achieved using an automated Nikon epifluorescent microscope equipped with an Okolab (
6
Fluorescence Imaging of Cryosections
7
Live-cell Imaging and Correlation Analysis
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