Imaging was performed on an Andor Revolution imaging system consisting of a Leica DMI6000B microscope, Yokogawa CSU-X1 spinning disk unit, Andor iXon camera, and an Andor FRAPPA unit for localized photoactivation. For photoactivation, a 445 nm laser beam was scanned every 5 s over the designated photoactivation region at a rate of 0.9 ms/μm2 at 145 nW power. Venus, EYFP, mTopaz, and AlexaFluor488 were excited with 515 nm light and emission was collected with 528/20 nm filter (Semrock). mCherry, mApple, and AlexaFluor594 were imaged with 594 nm light and emission was collected with 628/20 nm filter (Semrock). Images were acquired every 5 s with a 63× 1.4 NA oil immersion objective (Leica). Cells were maintained at 37 °C and 5% CO2 while imaging. Andor iQ software was used to control the photoactivation area and image acquisition.
Csu x1 spinning disk unit
Manufactured by Oxford Instruments
Sourced in United Kingdom
The CSU-X1 spinning disk unit is a laboratory instrument designed for high-speed confocal microscopy. It features a rotating Nipkow-type disk with thousands of pinholes that allows for rapid and efficient laser scanning of samples, enabling real-time imaging of live cells and other dynamic biological processes.
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Lab products found in correlation
Dmi6000b microscope, by Yokogawa (3 mentions)
Frappa unit, by Oxford Instruments (2 mentions)
Plan apo lambda, by Nikon (2 mentions)
Ti e pfs, by Nikon (2 mentions)
Ixon camera, by Oxford Instruments (1 mentions)
Na oil immersion objective, by Leica camera (1 mentions)
Tsc spe confocal microscope, by Leica camera (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Ixon electron multiplying charge coupled device camera, by Oxford Instruments (1 mentions)
897 emccd camera, by Oxford Instruments (1 mentions)
Nis elements, by Adobe (1 mentions)
Electron multiplying charge coupled device camera, by Oxford Instruments (1 mentions)
Ti r b inverted microscope, by Yokogawa (1 mentions)
Ultra 897bv, by Oxford Instruments (1 mentions)
9 protocols using csu x1 spinning disk unit
1
Quantitative Spatiotemporal Protein Dynamics
2
Confocal Microscopy Imaging Techniques
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Fixed samples were imaged using an inverted Leica TSC SPE confocal microscope. For representative images, a 60×/1.40NA oil immersion objective was used. For 4X scans a z-step size of 0.3 μm was used.
Live samples were imaged with an Andor revolution spinning disc confocal system, consisting of a Yokogawa CSU-X1 spinning disk unit and two Andor iXon3 DU-897-BV EMCCD cameras. A 60×/1.4NA oil immersion objective mounted on a Nikon Eclipse Ti microscope was used. Live imaging voxels sizes are 0.22 × 0.22 × 0.5 µm (60x/1.4NA spinning disc).
Live samples were imaged with an Andor revolution spinning disc confocal system, consisting of a Yokogawa CSU-X1 spinning disk unit and two Andor iXon3 DU-897-BV EMCCD cameras. A 60×/1.4NA oil immersion objective mounted on a Nikon Eclipse Ti microscope was used. Live imaging voxels sizes are 0.22 × 0.22 × 0.5 µm (60x/1.4NA spinning disc).
3
Optical Activation of Photosensitive Proteins
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Imaging and optical activation were performed using a spinning-disk confocal imaging system consisting of a Leica DMI6000B microscope with adaptive focus control, a Yokogawa CSU-X1 spinning-disk unit, an Andor iXon electron-multiplying charge-coupled device camera, a laser combiner with 445-, 488-, 515-, and 594-nm solid-state lasers, and an Andor FRAPPA unit for photoactivation of manually selected regions of the sample in real time, all controlled using Andor iQ2 software (Andor Technologies, Belfast, United Kingdom). For optical activation of iLID, the 445-nm laser was used at 5 μW and scanned across the selected region at a rate of 0.9 ms/μm2. This was performed once every 3–5 s. Laser wavelengths of 515 and 594 nm were used for excitation of Venus and mCherry, respectively. Emission filters were Venus 528/20 and mCherry 628/20 (Semrock). All images were acquired using a 63× oil immersion objective. A single confocal plane was imaged at a rate of 1 frame/3 s or 1 frame/5 s. All imaging was performed inside a temperature-controlled chamber held at 37°C. The chamber was also maintained at 5% CO2 during longer-duration experiments, that is, for samples kept on the microscope before and after treatment with Y-27632.
4
Live Sample Imaging with Confocal Microscopy
5
Imaging Ventral Midbrain Neurons
6
Live-cell TIRF Imaging of Gβγ/PIP3 Dynamics
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A spinning-disk XD confocal TIRF (total internal reflection) imaging system with a Nikon Ti-R/B inverted microscope, a Yokogawa CSU-X1 spinning disk unit (5000 rpm), an Andor FRAP-PA (fluorescence recovery after photobleaching and photoactivation) module, a laser combiner with 40−100 mW four solid-state lasers (with 445, 488, 515, and 594 nm wavelengths), and an iXon ULTRA 897BV back-illuminated deep-cooled EMCCD camera were used to capture time-lapse image series of live cells. In Gβγ translocation and PIP3 generation experiments, imaging was performed using a 60×, 1.4 NA (numerical aperture) oil objective. To examine the Gβγ translocation, GFP fluorescent tags on Gγ subunits (WTs and mutants) were imaged in every 1 s interval using 488 nm excitation−515 nm emission for 10 min. In PIP3 generation and PIP2 hydrolysis experiments, mCherry-tagged PIP3 and PIP2 sensors, Akt-PH and PH, were imaged using 594 nm excitation−630 nm emission red laser.
7
Anesthetizing and Imaging Live Worms
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10l of anesthetic (0.1% tricane and 0.01% tetramisole in 1X M9 buffer) was added to a 3% agar pad on a slide and 10-15 live worms were transferred to the drop of anesthetic.
A glass coverslip was slowly lowered to cover the samples and the coverslip edges were sealed with nail polish and allowed to dry before imaging. The images were obtained on a Nikon Ti-E-PFS inverted spinning-disk confocal microscope using a 60x 1.4NA Plan Apo Lambda objective. The microscope consists of a Yokowaga CSU-X1 spinning disk unit, a self-contained 4-line laser module (excitation at 405, 488,561, and 640nm), and an Andor iXon 897 EMCDD camera. Fluorescence intensities were quantified and image editing done using NIS-elements software.
A glass coverslip was slowly lowered to cover the samples and the coverslip edges were sealed with nail polish and allowed to dry before imaging. The images were obtained on a Nikon Ti-E-PFS inverted spinning-disk confocal microscope using a 60x 1.4NA Plan Apo Lambda objective. The microscope consists of a Yokowaga CSU-X1 spinning disk unit, a self-contained 4-line laser module (excitation at 405, 488,561, and 640nm), and an Andor iXon 897 EMCDD camera. Fluorescence intensities were quantified and image editing done using NIS-elements software.
8
Calcium Imaging of Sensory Nerve Endings
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Sensory nerve ending recordings were obtained from isolated dorsal hind paw skin preparations. The fur was removed with tape and the skin was gently dissected from the underlying tissue. Calcium imaging was performed on an inverted spinning disk confocal microscope (Nikon Ti; Yokogawa CSU-X1 Spinning Disk Unit, Andor, Belfast, Northern Ireland), equipped with a 20x air objective (NA 0.8), a 488 nm laser light and a EMCCD camera (iXon3 DU-897-BV, Andor). For image acquisition and instrument control, Andor iQ software was used. A z-stack of 11 frames (total thickness of 20-30 μm) was captured consecutively during the entire measurement at a speed of 0.25 Hz. Agonists were diluted in SIF and applied using a heated perfusion system (Multi Channel Systems, Reutlingen, Germany). Before image acquisition, the DRG samples were excited at 640 nm to detect WGA-647 + retrogradely labelled cell bodies.
9
Microglial Morphology Analysis in Dentate Gyrus
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Z-stacks of adjacent regions of the dentate gyrus were acquired with a spinning disk confocal microscope (Nikon Ti2-scope equipped with with an Andor Revolution CSU-X1 spinning disk unit and iXon DU-888 ultra EMCCD camera) using a 60x-oil immersion objective (NA 1.4). DAPI and Iba1 were excited with 10% 405 nm (15 mW) and 20% 561 nm (15mW) and bandpass filtered (425/45 and 685/40, respectively) before detection.
The images were segmented using the ImageJ Simple Neurite Tracing tool and Hessian-based analysis (Longair et al., 2011) . Microglial processes were traced in x, y, and z direction starting from the soma center. The starting point was used as the initial point for Sholl analysis. Analysis parameters were radius intervals of 2 µm on a standard axis and a linear profile. The automated calculation of the maximum branch length and Schoenen ramification index was done by the Sholl Analysis Tool in ImageJ (Norris et al., 2014; (link)Ferreira et al., 2014) . The resulting counts of 3 samples per animal were pooled and used for analysis.
The images were segmented using the ImageJ Simple Neurite Tracing tool and Hessian-based analysis (Longair et al., 2011) . Microglial processes were traced in x, y, and z direction starting from the soma center. The starting point was used as the initial point for Sholl analysis. Analysis parameters were radius intervals of 2 µm on a standard axis and a linear profile. The automated calculation of the maximum branch length and Schoenen ramification index was done by the Sholl Analysis Tool in ImageJ (Norris et al., 2014; (link)Ferreira et al., 2014) . The resulting counts of 3 samples per animal were pooled and used for analysis.
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