Time-lapse imaging was performed with an inverted microscope Nikon Ti-E installed into a thermostatically controlled chamber (Life Imaging Technologies) and equipped with a micro-incubator for thermal, CO2 and humidity control (OKOlab). The microscope was also equipped with an automated stage and a Yokogawa CSU-W1 spinning disk unit. Image acquisition was performed with an Andor Zyla 4.2 Plus camera, operated with Slidebook (ver.6.0.19). We performed fluorescence (60x lens, NA 1.4), phase contrast (10/20x objectives, NA 0.3/0.45) and differential interference contrast (DIC) imaging (20x lens, NA 0.45). 4D time-lapse was used for actin-labelled cell mounds (60x lens, NA 1.4) and for the pillar compression experiments. The latter combined DIC and confocal fluorescence modes (20x lens, NA 0.45). Typically, we acquired 12 images/h for >10h.
Csu w1
Manufactured by Yokogawa
Sourced in Japan
The CSU-W1 is a compact and versatile single-channel spectroscopic detector from Yokogawa. It features a high-performance spectrometer and a CCD detector, providing accurate spectral analysis capabilities in a compact and user-friendly design.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti, by Nikon (8 mentions)
Metamorph software, by Molecular Devices (7 mentions)
Metamorph, by Molecular Devices (7 mentions)
Axio observer z1, by Zeiss (6 mentions)
Ixon ultra 897, by Oxford Instruments (5 mentions)
Borealis, by Oxford Instruments (4 mentions)
Hcx pl apo 1.4 0 70na oil objective lens, by Leica (4 mentions)
Prime 95b, by Teledyne (4 mentions)
Ti2 inverted microscope, by Yokogawa (4 mentions)
Uplsapo 60xs2, by Olympus (4 mentions)
Csu x1, by Yokogawa (3 mentions)
Dmi8 microscope, by Yokogawa (3 mentions)
Nis element, by Nikon (3 mentions)
Orca fusion, by Hamamatsu Photonics (3 mentions)
Ix83 zdc, by Olympus (3 mentions)
Lu nv laser unit, by Nikon (3 mentions)
Zyla 4.2 scmos camera, by Oxford Instruments (3 mentions)
Ix83 fluorescence microscope, by Yokogawa (3 mentions)
Zyla 4, by Oxford Instruments (3 mentions)
Orca flash 4, by Hamamatsu Photonics (3 mentions)
109 protocols using csu w1
1
Time-lapse Imaging of Actin-labeled Cells
2
Confocal Microscopy Imaging Protocol
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Fixed samples were imaged using an inverted Leica SP5 confocal microscope. For representative images, a 60 × /1.40 N.A oil immersion objective was used. Live cell imaging was performed on a UltraView Vox spinning disc confocal microscope (Perkin Elmer Nikon TiE; Yokogawa CSU-X1 spinning disc scan head) with 60 × /1.40 N.A oil objective and equipped with a Hamamatsu C9100-13 EMCCD camera, or a 3I spinning disc confocal microscope (Zeiss AxioObserver Z1; Yokogawa CSU-W1 spinning disc scan head) with 63 × /1.40 N.A objective and equipped with a photometrics prime 95B scientific CMOS camera. Both spinning disc microscopes are equipped with a temperature-controlled environment chamber set at 26C for the experiments. Airyscan pictures were taken on fixed and stained neuroblasts using an inverted LSM880 Multiphoton or on live samples using a LSM900 point scanning laser confocal with Airyscan 2 on an inverted Axio Observer Z1 microscope stand.
3
Live Cell Imaging of DNA Repair
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Live cell imaging was performed with a Spinning Disk Confocal Microscope (CSU-W1, Yokogawa), with an electron multiplying charge device camera (ANDOR Zyla sCMOS) and a ×60/1.35 numerical aperture objective at 30 °C. Cells were mounted on agarose pads as described60 (link) and imaging recorded 15 z-sections with 0.5 µm spacing. Image acquisition was performed using Fiji (ImageJ)61 (link). Cells were grown in synthetic complete medium without uracil (SC-URA 2% raffinose), GFP-Yen1 was induced in a short burst with 30 min with Galactose at 2%, followed by addition of Glucose at 2%. DNA damage acute exposures (MMS 0.1% or 10 μg/ml Zeocin) lasted 15 min at room temperature following arrest of GFP-Yen1 expression. After the acute DNA damage, cells were washed once with fresh SC-URA 2% glucose and held for 30 min at 30 °C in this medium, whereas aliquots were removed at the indicated times. Cells showing an accumulation of spots were measured at maximum projection of the GFP channel. Statistical analysis was performed using Fisher's exact test to determine the level of significance between two categories and χ2 to compare more than two categories and consistency between trials.
4
Confocal Imaging of Fission Yeast
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Confocal fluorescence imaging was performed on an IX83 equipped with a UPALXAPO60XO/1.42 NA oil objective lens (Olympus), a UPALXAPO100XO/1.45 NA oil objective lens (Olympus), a Zdrift compensator system (IX3-ZDC2, Olympus), a sCMOS camera (Orca-Fusion BT; Hamamatsu Photonics), and a spinning disk confocal unit (CSU-W1; Yokogawa Electric Corp.). For fission yeast time-lapse imaging, a CellASIC ONIX2 Microfluidic System (CAX2-S0000, Millipore) and a CellASIC ONIX2 Manifold XT (CAX2-MXT20, Millipore) were used. The microscope was controlled by MetaMorph software (Molecular Devices). A 488 nm laser was used for excitation of mNeonGreen.
An excitation dichroic mirror (DM405/488/561/640) and emission filter (525/50 for mNeonGreen) were used for confocal fluorescence imaging (Yokogawa Electric Corp.). Fluorescence images were analyzed using Fiji/ImageJ 44 .
An excitation dichroic mirror (DM405/488/561/640) and emission filter (525/50 for mNeonGreen) were used for confocal fluorescence imaging (Yokogawa Electric Corp.). Fluorescence images were analyzed using Fiji/ImageJ 44 .
5
Seedling Live-Cell Imaging Protocols
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All other live cell imaging was conducted using CSU-X1 or CSU-W1 Yokogawa spinning disk head fitted to a Nikon Ti-E inverted microscope. For seedling imaging, 3-d-old etiolated hypocotyls or roots were mounted in water under an agarose pad. For BFA-treatments, seedlings were treated with BFA (50 μM) in ½ MS media with 1% sucrose. FM4-64 straining was performed for 10 min with 2 µM FM4-64.
6
Spinning Disk Microscopy Technique
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Spinning disk images were acquired using a 60×/1.4NA objective on a Zeiss Axioobserver Z1 microscope equipped with a cMOS camera coupled to a Yokogawa CSU-W1 spinning disk. Metamorph Software (Universal Imaging) was used to collect data.
7
Whole-Brain Neural Activity Imaging in C. elegans
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To capture whole brain neural activity in a freely behaving C.elegans, we combined a spinning disk confocal inverted microscope (Nikon Ti-U and Yokogawa CSU-W1, Japan) with a customized upright light path for worm tracking. Fluorescence signals, emitted from neurons at different depths in a head ganglion, were collected by a high NA objective (40X, NA = 0.95, Nikon Plan Apo), driven by a high-precision scanner (PI P721.CDQ). The measured lateral resolution is 0.30 μm/pixel; the scanning step along Z-axis is 1.50 μm. An imaging volume comprises 18 two-channel fluorescence images recorded at 100 fps by two sCMOS cameras (Andor Zyla 4.2, England) simultaneously. Our imaging system thus has a volume rate ≈ 5 Hz. Reducing the volume rate by increasing the inter-volume interval is not critical for CeNDeR, since both our training and inference methods are sequence-independent. A customized infrared LED ring (850 nm) was mounted above a worm, and dark-field images of worm behaviors were captured by an upright light path and recorded at 25 fps by a USB-3.0 camera (Basler acA2000–165umNIR).
8
Imaging Yap1-Wwtr1 Mutant Embryos
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Embryos from incrosses of yap1;wwtr1 double heterozygous animals were injected with 10 pg of H2A-mCherry and lyn-EGFP mRNA each at the one-cell stage. At 90% epiboly, 6-10 embryos were dechorionated and embedded laterally in 0.5% LMA on a glass-bottomed dish to be imaged under the spinning disk system (Yokogawa CSU-W1). A z-stack of 41 slices, 0.5 µm thick, were acquired per embryo every 2 minutes overnight in an environmental chamber set at 28.5°C. At most 8 embryos were imaged in a single sitting. Embryos were genotyped the next day. All mutants are yap1 -/-;wwtr1 -/-, while siblings are not yap1 -/-or wwtr1 -/-.
9
Imaging Caenorhabditis elegans Embryogenesis
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All imaging was carried out using a Leica DMi8 microscope outfitted with a spinning disk confocal unit–CSU-W1 (Yokogawa) with Borealis (Andor), dual iXon Ultra 897 (Andor) cameras, and a 100x HCX PL APO 1.4–0.70NA oil objective lens (Leica). (Molecular Devices) imaging software was used for controlling image acquisition. The 488nm and 561nm channels were imaged simultaneously with 1um Z-spacing.
Ex utero live imaging used the following parameters: 1μm Z spacing, 16 focal planes, 100ms exposure, 10s interval. Imaging was carried out by dissecting worms in 3ul egg salt buffer (118mM NaCl, 48mM KCl, 2mM CaCl2, 2mM MgCl2, and 0.025 mM of HEPES, filter sterilized before HEPES addition) on a coverslip before mounting onto a 2% agarose pad (diluted in egg salt buffer) on a microscope slide.
In utero live imaging used the following parameters: 1 μm Z spacing, 20 μm stacks, 80s exposure, 20s interval. Imaging was carried out by placing adult worms in 1.5 μl of M9 mixed with 1.5 μl of 1 μm polysterene microspheres (Polysciences Inc.) on a coverslip which was mounted carefully onto a 5% agarose pad.
Ex utero live imaging used the following parameters: 1μm Z spacing, 16 focal planes, 100ms exposure, 10s interval. Imaging was carried out by dissecting worms in 3ul egg salt buffer (118mM NaCl, 48mM KCl, 2mM CaCl2, 2mM MgCl2, and 0.025 mM of HEPES, filter sterilized before HEPES addition) on a coverslip before mounting onto a 2% agarose pad (diluted in egg salt buffer) on a microscope slide.
In utero live imaging used the following parameters: 1 μm Z spacing, 20 μm stacks, 80s exposure, 20s interval. Imaging was carried out by placing adult worms in 1.5 μl of M9 mixed with 1.5 μl of 1 μm polysterene microspheres (Polysciences Inc.) on a coverslip which was mounted carefully onto a 5% agarose pad.
10
Microscopy Techniques for Gut Architecture
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Different microscopes were utilized based on the study. Bright‐field images of the µGut architecture and time‐course images of Caco‐2 culture were recorded using an inverted phase‐contrast microscope (Nikon Eclipses Ti ; Nikon with EvolveTM camera). The morphology of µGut structure and microvilli were imaged by a spinning disk confocal microscopy comprising an inverted microscope (Ti‐E, Nikon), a spinning disk scan head (CSU‐W1; Yokogawa), an sCMOS camera (Prime95B; Teledyne Photometrics) and a laser system (iLaunch; GATACA Systems). The µGut samples were visualized with CFI plan Apo objective under laser excitation 404/561/642 nm and scanned with Z‐series at step size 1 µm. The images were processed with MetaMorph (Molecular Devices), IMARIS (Bitplane Scientific software), and Image J. 3D mapping of the Caco‐2 cells and bacteria as well as cell and villi height measurements were performed using IMARIS. All image‐based quantification for cell signaling pathways were performed using an ImageJ script developed in‐house. For cell signaling quantification, all the samples were normalized against the hypoxic control, except for E‐cadherin, which was plotted without normalization.
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