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Csu w1 spinning disk

Manufactured by Nikon
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The Nikon CSU-W1 is a spinning disk confocal microscope unit. It is designed to provide high-speed, high-resolution imaging of live cells and tissues. The CSU-W1 uses a rapidly rotating disk with thousands of pinholes to scan the sample, allowing for the acquisition of images at high frame rates.

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Lab products found in correlation

Perfect focus 2, by Nikon (4 mentions) Fibronectin, by Merck Group (3 mentions) Nis elements software, by Nikon (3 mentions) Eclipse ti 2 stand, by Yokogawa (2 mentions) Andor888 em ccd camera, by Nikon (2 mentions) Oil objective lens, by Nikon (2 mentions) Gfp trap beads, by Proteintech (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Ixon 888 emccd camera, by Oxford Instruments (2 mentions) 1.45 na oil objective lens, by Nikon (2 mentions) Zyla 4.2mpx scmos camera, by Oxford Instruments (2 mentions) 1.3 na objective lens, by Nikon (2 mentions) Spectra x led illumination system, by Lumencor (2 mentions) Boldline temperature co2 controller, by Okolab (2 mentions) μ slide 8 well, by Ibidi (2 mentions) Prime 95b, by Nikon (2 mentions) An objective heater, by Bioptechs (2 mentions) Tetraspeck fluorescent microspheres size kit, by Thermo Fisher Scientific (1 mentions) Orca fusion camera, by Nikon (1 mentions) Lsm meta, by Zeiss (1 mentions)

22 protocols using csu w1 spinning disk

1

Comparing Confocal-Like Imaging Techniques

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Imaging at near confocal resolution was carried out with an Aurox Clarity laser-free microscopy system (Abingdon, Oxfordshire, OX14 3DB, UK) (19 (link)), employing a Nikon Eclipse TE 2000-S inverted microscope with an APO LWD 40× 1.15 NA water immersion lens. The 50% point spread was 0.7 and 1.6 microns in the lateral and vertical directions, respectively, as determined with a TetraSpeck Fluorescent Microspheres Size Kit (Invitrogen). This compares to 0.6 and 2.9 microns for a 40× 0.95 NA air lens employed on a standard spinning disc microscope (Nikon CSU-W1 spinning disk with Hamamatsu Orca Fusion camera, Nikon CSU-W1 SoRa: Quantitative Light Microscopy Core Facility, UTSouthwestern). Equivalent images obtained from the same slide (FluoCells prepared slide #3, Invitrogen) with these two imaging systems were provided previously (29 ).
Deisl C., Moe O.W, & Hilgemann D.W. (2024). Constitutive Plasma Membrane Turnover in T-REx293 cells via Ordered Membrane Domain Endocytosis under Mitochondrial Control. bioRxiv.
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2

Quantifying Muscle Fiber Characteristics

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Imaging was performed in the UMB Confocal Microscopy Core on an LSM-DUO (Carl Zeiss, Jena, Germany), Nikon CSU-W1 spinning disk (Nikon Instruments, Melville, NY) or an LSM-Meta (Carl Zeiss) confocal microscope. We used ImageJ or FIJI to measure the relative areas of each cross-section that were occupied by either mouse or human muscle fibers, as well as to quantify human fiber number, minimum Feret’s diameter, compaction (measured as fiber-to-fiber distances), and central nucleation manually, as described10 (link)
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O’Neill A., Martinez A.L., Mueller A.L., Huang W., Accorsi A., Kane M.A., Eyerman D, & Bloch R.J. (2024). Optimization of Xenografting Methods for Generating Human Skeletal Muscle in Mice. Cell Transplantation, 33, 09636897241242624.
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3

Confocal Microscopy with Live-SR

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Confocal images were captured using a 100× Plan-Apo 1.45 numerical aperture (NA) oil immersion objective on a Nikon TiE microscope attached to a CSU-W1 spinning-disk confocal head. For images in Figs. 4A and 7A, Live-SR module (Gataca Systems) on the spinning-disk microscope was engaged during imaging and post processing of the images was carried out using Live-SR algorithm.
Pan M., Chew T.W., Wong D.C., Xiao J., Ong H.T., Chin J.F, & Low B.C. (2020). BNIP-2 retards breast cancer cell migration by coupling microtubule-mediated GEF-H1 and RhoA activation. Science Advances, 6(31), eaaz1534.
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4

BMH-21 Treatment of Mouse Embryos

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E8.5 embryos from C57Bl/6 pregnant dam were harvested and cultured in 50% rat serum in DMEM-F12 media in 20% CO2 at 37C. After 30 minutes of normalization in the culture conditions, the embryos were treated with 1 μM BMH-21 for 8 hours. The controls were treated with DMSO. After 8 hours, the embryos were then fixed and immunostained. Further, the embryos were cryo-sectioned and imaged using Nikon Ti2 microscope with CSU-W1 Spinning Disk. All images for each stain were individually acquired with the same settings, and brightness and contrast were adjusted the same throughout.
Dash S., Lamb M.C., Lange J.J., McKinney M.C., Tsuchiya D., Guo F., Zhao X., Corbin T.J., Kirkman M., Delventhal K., Moore E.L., McKinney S., Shiang R, & Trainor P.A. (2023). rRNA transcription is integral to phase separation and maintenance of nucleolar structure. PLOS Genetics, 19(8), e1010854.
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5

Imaging and Analysis of Cochlear Tissues

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Images of cochlear whole mounts and organotypic cultures were acquired using either a Yokogawa CSU-W1 spinning disk on a Nikon Ti2 microscope with a Hamamatsu Flash 4.0 V3 camera operated by NIS-Elements or a Nikon A1 confocal microscope, using ×100 or ×60 lenses. Exposure times were set to ensure high signal-to-noise ratio and no saturation in the image. Gain and offset adjusting were performed to ensure that no saturated or undersaturated pixels were present. Identical capture and analysis conditions were used for each experimental and control tissue.
Images were processed and three-dimensional renderings were generated using NIS-Elements and Imaris. Nuclear volumes were measured on the basis of DAPI fluorescence using Imaris, as detailed in ref. 2 (link).
García-Añoveros J., Clancy J.C., Foo C.Z., García-Gómez I., Zhou Y., Homma K., Cheatham M.A, & Duggan A. (2022). Tbx2 is a master regulator of inner versus outer hair cell differentiation. Nature, 605(7909), 298-303.
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6

Immunofluorescence Imaging of Cells

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Cells were seeded in 4-well Lab Tek II Chamber Slides (Thermo Fisher Scientific). The following day, cells were fixed for 15 minutes with 4% paraformaldehyde, washed, and incubated in blocking buffer for 1 hour (1× PBS with 1% BSA and 0.3% Triton X-100). Blocking buffer was aspirated and cells were incubated with primary antibody overnight in the dark at 4°C. The following day, cells were washed, incubated with fluorophore-conjugated secondary antibody for 1 hour at room temperature in the dark, washed, then mounted using ProLong Gold Antifade reagent with DAPI (Cell Signaling Technology, Danvers, MA). Slides were analyzed using a Nikon Ti microscope with a CSU-W1 spinning disk confocal using a 100×/1.4 Plan Apo VC objective (Nikon Imaging Center, UCSF). Images were acquired on MicroManager software and analyzed using Fiji software(26 ,27 ).
Neel D.S., Allegakoen D.V., Olivas V., Mayekar M.K., Hemmati G., Chatterjee N., Blakely C.M., McCoach C.E., Rotow J.K., Le A., Karachaliou N., Rosell R., Riess J.W., Nichols R., Doebele R.C, & Bivona T.G. (2018). Differential subcellular localization regulates oncogenic signaling by ROS1 kinase fusion proteins. Cancer research, 79(3), 546-556.
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7

Visualizing Adenovirus Protein Colocalization

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Appropriate cell lines were plated on culture dishes (μ-Dish 35 mm, glass bottom, ibidi) that were coated with 1% (v/v) fibronectin (Sigma Aldrich) for 40 min, infected with varying HAdV5 strains and prepared at 24 or 48 hpi for imaging of proteins of interest. Images were recorded on an inverted confocal spinning-disk microscope (Nikon Eclipse Ti-2 stand; Yokogawa CSU-W1 spinning disk; 2x Andor888 EM-CCD camera; Nikon 100x oil-immersion numerical aperture (NA) 1.49 objective) equipped with a heating chamber at 37°C and 5% (v/v) CO2. Images were recorded using the Nikon NIS-Elements software. Further image processing was performed in Fiji [51 (link)]. Colocalization quantification of fluorescence signal was performed using the Fiji JaCoP colocalization plugin [52 (link)]. In JaCoP, Mander’s colocalization coefficients were based on a background threshold, that is calculated by the plugin based on the input images to remove noise contribution within the image.
Pfitzner S., Hofmann-Sieber H., Bosse J.B., Franken L.E., Grünewald K, & Dobner T. (2020). Fluorescent protein tagging of adenoviral proteins pV and pIX reveals ‘late virion accumulation compartment’. PLoS Pathogens, 16(6), e1008588.
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8

Subcellular Organelle Colocalization Imaging

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L4 animals carrying dzSi3 and single-copy insertions of GFP subcellular compartment markers were imaged using a Plan-Fluor Nikon 60x/1.4x objective on a Nikon CSU-W1 Spinning Disk. Single planes from a confocal image stack in which both the catp-8 and organellar reporters were clearly visible were manually thresholded, then analyzed using the Correlation Plot application in Metamorph 7.7 to establish a Pearson’s Correlation Coefficient for each image pair.
Tang L.T., Trivedi M., Freund J., Salazar C.J., Rahman M., Ramirez-Suarez N.J., Lee G., Wang Y., Grant B.D, & Bülow H.E. (2021). The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning. PLoS Genetics, 17(7), e1009475.
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9

In Vitro Reconstitution of Spindly-Mediated Filament Formation

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Filamentation was performed in 10 μl of 50 mM Hepes, pH 7.5, 10 mM KCl, 1 mM MgCl2 containing 2 μM of mCherry-RZZ and 8 μM of farnesylated SpindlyFL isolated from insect cells. Reaction was incubated at the indicated conditions for 1 hour and filaments were imaged in #1.5 glass bottom 96-well plates (Cellvis) on an Andor CSU-W1 spinning disk (50 µm disk) with 100x 1.45 NA oil objective lens (Nikon). Where indicated, filament reaction was induced in the presence of GFP-Spindly beads or empty beads at 30°C. Before imaging, beads were diluted in 100 μl of buffer. To immobilize GFP-Spindly in agarose beads, HeLa cells expressing GFP-Spindly were arrested in nocodazole and collected by mitotic shake-off. Cells were lysed in 50 mM Hepes, pH 7.5, 10 mM KCl, 1 mM MgCl2 supplemented with 1% NP-40 and protease inhibitor cocktail (Roche). Lysates were sonicated and centrifuged for 10’ at 20.000 x g at 4°C. Supernatants were collected and incubated with GFP-Trap beads (Chromotek) for 2 hours at 4°C and washed 5 times in the same buffer without NP-40.
Sacristan C., Ahmad M.U., Keller J., Fermie J., Groenewold V., Tromer E., Fish A., Melero R., Carazo J.M., Klumperman J., Musacchio A., Perrakis A, & Kops G.J. (2018). Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis. Nature cell biology, 20(7), 800-810.
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10

In Vitro Reconstitution of Spindly-Mediated Filament Formation

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Filamentation was performed in 10 μl of 50 mM Hepes, pH 7.5, 10 mM KCl, 1 mM MgCl2 containing 2 μM of mCherry-RZZ and 8 μM of farnesylated SpindlyFL isolated from insect cells. Reaction was incubated at the indicated conditions for 1 hour and filaments were imaged in #1.5 glass bottom 96-well plates (Cellvis) on an Andor CSU-W1 spinning disk (50 µm disk) with 100x 1.45 NA oil objective lens (Nikon). Where indicated, filament reaction was induced in the presence of GFP-Spindly beads or empty beads at 30°C. Before imaging, beads were diluted in 100 μl of buffer. To immobilize GFP-Spindly in agarose beads, HeLa cells expressing GFP-Spindly were arrested in nocodazole and collected by mitotic shake-off. Cells were lysed in 50 mM Hepes, pH 7.5, 10 mM KCl, 1 mM MgCl2 supplemented with 1% NP-40 and protease inhibitor cocktail (Roche). Lysates were sonicated and centrifuged for 10’ at 20.000 x g at 4°C. Supernatants were collected and incubated with GFP-Trap beads (Chromotek) for 2 hours at 4°C and washed 5 times in the same buffer without NP-40.
Sacristan C., Ahmad M.U., Keller J., Fermie J., Groenewold V., Tromer E., Fish A., Melero R., Carazo J.M., Klumperman J., Musacchio A., Perrakis A, & Kops G.J. (2018). Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis. Nature cell biology, 20(7), 800-810.
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