Cells were grown at 37 °C in TH to an OD600nm of 0.3, transferred to microscope slides, and observed at 37 °C on an Olympus IX81 microscope equipped with a Yokogawa CSU-X1 confocal spinning disk unit. Image acquisition was performed using a UAPON 100× (N.A. 1.49) oil immersion objective and AndorTMiXon Ultra Electron Multiplying Charge Coupled Device (EMCCD) camera. Epifluorescence confocal of GFP signal (488 nm excitation and 520/28 nm emission) and Differential Interference Contrast (DIC) transmission imaging were collected using IQ software (AndorTM), and further analyzed using Volocity (Perkin ElmerTM). In addition to the specific GFP channel, images were acquired in the red emission channel (561 nm excitation and 617/73 nm emission) to estimate the background autofluorescence signal. In each frame (total of five) and for each cell, the maximal fluorescence intensities were measured and the mean maximal intensity of GFP for each sample was calculated after substraction of the autofluorescence levels. The error bars correspond to the standard deviation of the analysis of the five independent frames acquired for each sample.
Csu x1 spinning disk confocal unit
Manufactured by Yokogawa
Sourced in Japan
The CSU-X1 is a spinning-disk confocal unit designed for high-speed, high-resolution imaging in biological research. It utilizes a disk with thousands of pinholes to simultaneously scan multiple points of a sample, providing fast image acquisition. The CSU-X1 is capable of capturing images at up to 2,000 frames per second, making it suitable for live-cell imaging and dynamic processes.
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Lab products found in correlation
Eclipse ti inverted microscope, by Nikon (2 mentions)
Ichrome mle l multilaser engine, by Toptica (2 mentions)
Dmi6000 microscope, by Leica (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Escalab 250xi, by Thermo Fisher Scientific (2 mentions)
Ix81 microscope, by Yokogawa (1 mentions)
Co2 independent medium, by Thermo Fisher Scientific (1 mentions)
Draq7, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Axio observer inverted microscope, by Zeiss (1 mentions)
Orca er cooled ccd camera, by Hamamatsu Photonics (1 mentions)
Ixon du 897 emccd camera, by Oxford Instruments (1 mentions)
Orca er camera, by Hamamatsu Photonics (1 mentions)
Ix71 inverted microscope, by Olympus (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Orca flash4.0 v3 digital scmos camera, by Hamamatsu Photonics (1 mentions)
17 protocols using csu x1 spinning disk confocal unit
1
Quantifying GFP Fluorescence in Single Cells
2
Single-molecule dynamics of key signaling proteins
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An Eclipse Ti Inverted Microscope (Nikon) with a TIRF system and Evolve EMCCD Camera (Photometrics) were used for live-cell imaging. TIRF microscopy was performed with a 100× TIRF objective with a numerical aperture of 1.49 (Nikon) and an iChrome MLE-L multilaser engine as a laser source (Toptica Photonics). Immunofluorescent imaging was also acquired in an Eclipse Ti Inverted Microscope (Nikon) with CSU-X1 confocal spinning disk unit (Yokogawa).
Time-lapse single-molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2, and N-WASP-mEos3.2 were performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of individual molecules was controlled by 405 nm laser illumination to be about ~0.5/µm2. Far-red channel (ex=647 nm, em>655 nm) were acquired before single-molecule recording to localize mobile and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of Eos molecules were visualized and recorded by EMCCD with 20 frames per s video rate. Each movie contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to calculate photobleach rate, acquired at the same microscopic setup.
Time-lapse single-molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2, and N-WASP-mEos3.2 were performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of individual molecules was controlled by 405 nm laser illumination to be about ~0.5/µm2. Far-red channel (ex=647 nm, em>655 nm) were acquired before single-molecule recording to localize mobile and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of Eos molecules were visualized and recorded by EMCCD with 20 frames per s video rate. Each movie contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to calculate photobleach rate, acquired at the same microscopic setup.
3
Single-Molecule Imaging of Signaling Proteins
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An Eclipse Ti inverted microscope (Nikon) with a TIRF system and Evolve EMCCD camera (Photometrics)
was used for live cell imaging. TIRF microscopy was performed with a 100x TIRF objective with a numerical aperture of 1.49 (Nikon) and an iChrome MLE-L multilaser engine as a laser source (Toptica Photonics). Immunofluorescent imaging was also acquired in an Eclipse Ti inverted microscope (Nikon)
with CSU-X1 confocal spinning disk unit (Yokogawa).
Time-lapse single molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2 and NWASP-mEos3.2 were performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of individual molecules was controlled by 405 nm laser illumination to be about ~0.5 / µm 2 . Far-red channel (ex =647 nm, em > 655 nm) were acquired before single molecule recording to localize mobile and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of Eos molecules were visualized and recorded by EMCCD with 20 frame per second video rate. Each movie contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to calculate photobleaching rate, acquired at the same microscopic set up.
was used for live cell imaging. TIRF microscopy was performed with a 100x TIRF objective with a numerical aperture of 1.49 (Nikon) and an iChrome MLE-L multilaser engine as a laser source (Toptica Photonics). Immunofluorescent imaging was also acquired in an Eclipse Ti inverted microscope (Nikon)
with CSU-X1 confocal spinning disk unit (Yokogawa).
Time-lapse single molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2 and NWASP-mEos3.2 were performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of individual molecules was controlled by 405 nm laser illumination to be about ~0.5 / µm 2 . Far-red channel (ex =647 nm, em > 655 nm) were acquired before single molecule recording to localize mobile and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of Eos molecules were visualized and recorded by EMCCD with 20 frame per second video rate. Each movie contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to calculate photobleaching rate, acquired at the same microscopic set up.
4
Anthrax toxin imaging in intestinal enteroids
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LFNRod (Invivogen, tlrl-rod) was reconstituted in endotoxin-free water at a concentration of 500 μg/ml and stored at −80°C in aliquots until used. PA was expressed and purified from BL21(DE3)/pET22b-PA (Addgene) as previously described [74 (link)] with an end concentration of 300 to 500 μg/ml stored at −80°C in aliquots until used. 2D-grown enteroids were gently washed in PBS and 100 μl CO2-independent medium (Thermo Fischer Scientific) containing 10% FBS (Thermo Fischer Scientific), 1×Pen-Strep, 1×GlutaMAX (Thermo Fischer Scientific), and 1:1,000 diluted DRAQ7 (Thermo Fischer Scientific) was added to the cells. Medium was further supplemented with 5 μg/ml LFNRod and 10 μg/ml PA when indicated. Cells were incubated at room temperature (Figs 2 and S3 ) or at 37°C (S3 Fig ) and imaged every 5 min. The imaging set up consisted of a 20× objective on an Axiovert 200 m (Zeiss) microscope, an X/Y motorised stage (Ludl), a CSU- X1 spinning disk confocal unit (Yokogawa) (488 nm laser with ET 525/50 filter or 640 nm laser with ET 700/75 filter), and an Evolve 512 EMCCD camera (Photometrics) (Figs 2C and S3 ) or a 40× objective on an AxioObserver Z1 microscope (Zeiss), an X-cite light system (EXFO), fluorescence filter sets 38 HE and 50 (Zeiss), and an ORCA-Fusion BT Digital CMOS camera (Hamamatsu) (Fig 2A and 2B ).
5
Spinning-Disk Confocal Microscopy Workflow
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All images were acquired with a Mariana system (Intelligent Imaging Innovations, Denver, CO) based on a Zeiss Axio-Observer inverted microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) equipped with a CSU-X1 spinning-disk confocal unit (Yokogawa Electric, Tokyo, Japan), a piezo-driven Z-translation, and linear encoded X&Y translations and controlled with SlideBook V5.0 (Intelligent Imaging Inc., Denver, CO). Excitation wavelengths were 405, 488, 561, and 640 nm (lasers were from Cobolt, Solna, Sweden); the emission filters were 452/45, 525/50, 607/36, and 680-nm long-pass (Semrock, Rochester, NY). Exposure times and laser settings for each experiment are outlined above.
6
Multimodal Microscopy Imaging Protocol
7
Real-Time Imaging of Protein Trafficking in HeLa Cells
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HeLa cells were seeded onto 18 mm-diameter glass coverslips, 1 day before transfection. Twenty hours after transfection with the TNFα-SBPEGFP RUSH plasmid (Boncompain et al., 2012 (link)), coverslips were transferred into a Chamlide chamber, filled with pre-warmed DMEM medium (Invitrogen). At time 2 min, medium was removed and D-biotin (Sigma-Aldrich) at 40 μM final was introduced in the chamber. Time-lapse acquisitions were done at 37°C in a thermostat-controlled chamber. Fluorescent images were sequentially acquired every 40 s for 60 min using a HCX APO 1.3 glycerol 63 X objective and Metamorph software (Molecular Device). We used a LEICA DMI8 microscope (LEICA MICROSYSTEMS) equipped with a CSU-X1 spinning-disk confocal unit (Yokogawa Electric Corporation) and an ORCA -Flash4.0 V3 Digital sCMOS camera (Hamamatsu Photonics) in a controlled environment box (37°C and 5% CO2, PECON). The microscopy system was equipped with a laser combiner (Errol) comprises of a 488 nm (KVANT) and a 561 nm (Oxxius LBX) laser line. GFP (resp. mCherry/mRFP) emission light were collected with a stringent single bandpass filter 525/50-25 (resp. 620/60-25). The microscopy system was driven by Metamorph (Molecular Devices).
8
Characterization of Au/CeO2 Nanoparticles
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Hydrodynamic size and zeta potential were measured in the deionized water on Nano-ZS ZEN3600 (Malvern). The morphology of nanoparticles was characterized by the transmission electron microscope (Hitachi H-7000FA) and high-resolution transition electron microscope (HR-TEM) (Jeol2100f). Jed2300 was used to observe the element distribution of Au/CeO2. X-ray powder diffraction (XRD) patterns were recorded by Bruker D8 Advance. X-ray photoelectron spectroscopy (XPS) spectra were performed by ESCALAB 250Xi (Thermo Fisher). Thermogravimetric analysis (TGA) was carried out by TA TGA55. Temperature-programmed reduction (TPR) analysis was measured by AutoChem1 II 2920. The N2 adsorption/desorption isotherm was performed by ASAP 2460 3.01 (Micromeritics, USA), and the surface area and pore size were calculated using the Brunauer-Emmett-Teller (BET) model. Inductively coupled plasma mass spectrometry (ICP-MS, ThermoFisher iCAP-TQ) was employed to measure the retained concentrations of Au/CeO2 or Au/CeO2@HA in the colon tissues.
The cellular internalization of rhodamine B-labeled nanoparticles was evaluated using a Nikon Ti–U microscopy equipped with a CSU-X1 spinning-disk confocal unit (Yokogawa) and an EM-CCD camera (iXon+; Andor).
The cellular internalization of rhodamine B-labeled nanoparticles was evaluated using a Nikon Ti–U microscopy equipped with a CSU-X1 spinning-disk confocal unit (Yokogawa) and an EM-CCD camera (iXon+; Andor).
9
Imaging Mounted Ovaries Using Confocal Microscopy
10
High-Resolution Imaging of Live Cells
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Approximately 1 × 105 U373 cells were plated 16 h before imaging on 25-mm (diameter) no. 1.5 glass coverslips. The imaging medium was phenol red–free DMEM supplemented with 10% FCS and 20 mM HEPES (pH 7.4). For imaging (Kural et al., 2012 (link)), the coverslips were placed on a temperature-controlled 5% CO2 humidified chamber (20/20 Technology, Wilmington, NC) mounted on the piezo-electric driven stage of a Mariana imaging system (Intelligent Imaging Innovations, 3I, Denver, CO) based on an Axiovert 200M inverted microscope (Carl Zeiss, Thornwood, NY), a CSU-X1 spinning-disk confocal unit (Yokogawa Electric Corporation, Tokyo, Japan), a spherical aberration-correction device (SAC; Infinity Photo-Optical, Boulder, CO), and a 63× objective lens (Plan-Apochromat, NA 1.4, Carl Zeiss). The SAC was placed between the oil-based objective lens and the camera to resolve the spherical aberration introduced by the refractive index mismatch between living cells and the glass optics. Three-dimensional time series were obtained using Slidebook 5 (Intelligent Imaging Innovations).
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