Immunofluorescence images of vascular networks in the 3D angiogenesis device were recorded using an Olympus DSU-IX81 Spinning Disc Confocal Microscope equipped with an EMCCD camera (iXon X3, Andor). Z-stack images were acquired with a slice thickness of 1 µm. Time-lapse images of angiogenesis were acquired during a time period of 48 hr with an interval of 30 min after initial cell seeding. Cells were kept at 37°C using a Tokai Hit incubator chamber placed on top of the microscope stage. Migration trajectories of individual invading tip cells were tracked manually using MTrackJ plugin of ImageJ. Orientation angle of tip cell invasion was calculated using coordinates of the starting and end points of an individual cell invasion trajectory. Fluorescence images of EC angiogenesis with internalized GNR-LNA probes were also acquired using confocal microscopy at 6, 12 and 18 hr after cell seeding and analyzed with ImageJ to determine fluorescence intensity of LNA probes.
Ixon x3
Manufactured by Oxford Instruments
Sourced in Japan, United Kingdom
The IXon X3 is a high-performance EMCCD (Electron Multiplying Charge-Coupled Device) camera from Oxford Instruments. The camera is designed for low-light imaging and spectroscopy applications, providing high sensitivity and fast frame rates.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti, by Nikon (5 mentions)
Csu x1, by Oxford Instruments (4 mentions)
Plan apochromat na 1.43 objective lens, by Nikon (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Ix81 microscope, by Olympus (2 mentions)
α plan fluar, by Zeiss (2 mentions)
Tirf illuminator, by Zeiss (2 mentions)
Axiovert 200m microscope, by Zeiss (2 mentions)
Csu x1, by Yokogawa (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Illustrator cs6, by Adobe (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Axiocam, by Zeiss (1 mentions)
Xl s1 incubator, by Zeiss (1 mentions)
Yoyo 1, by Thermo Fisher Scientific (1 mentions)
3d sim system, by Nikon (1 mentions)
Plan apochromat na 1.4 objective lens, by Nikon (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
A1 confocal laser microscope, by Nikon (1 mentions)
Csu22, by Yokogawa (1 mentions)
18 protocols using ixon x3
1
3D Angiogenesis Imaging and Tip Cell Tracking
2
Quantifying Subcellular YAP Localization
3
Single-Molecule Imaging in Microscopy
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Single-molecule images were acquired using an Axiovert 200M microscope with a TIRF illuminator (Zeiss, UK) and incorporating a 100 x oil-immersion objective (α-Plan-Fluar, NA = 1.45; Zeiss, UK) and an EMCCD (iXon X3; Andor, UK). Samples were illuminated with a 638 nm laser (100 mW, Vortran) fed into the microscope via a polarisation maintaining triple laser combiner (Oz Optics) . Alternatively, the 640 nm lines of a Vortran Combiner or of an Andor Revolution Laser Combiner were used. A wrap-around incubator (Pecon XL S1) was used to maintain a constant temperature of 37°C. The field of view of each channel was 80 × 30 μm. Data were acquired at 20 Hz for 30 s. Images were saved in HDF5 format for subsequent processing using custom-designed software. All Single-Molecule time series data were analysed using the multidimensional analysis software described previously (Rolfe et al., 2011 (link)).
4
Live-cell imaging of mCherry-H2B and GalT-GFP in HeLa cells
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For live-cell imaging, stable HeLa cells expressing mCherry–histone H2B and GalT-GFP were plated in 35-mm glass-bottom dishes (glass diameter of 14 mm, glass thickness of 1.5; MatTek Corporation). Time-lapse microscopy was performed beginning at 8 or at 24 h after transfection using a 40× oil objective (NA 1.3), no binning, on an inverted confocal microscope (LiveScan Swept Field; Nikon), Piezo Z stage (Nano-Z100N; Mad City Labs, Inc.), and an electron-multiplying charge-coupled device camera (512 × 512; iXon X3; Andor Technology). The microscope was equipped with an environmental chamber heated to 37°C with 5% CO2. Images were acquired with NIS-Elements Version 4.0 acquisition software every 2 min using a 0.2-s exposure at 0.5-µm increment sizes with a slit size of 50 µm for 15–20 h. Images were viewed and analyzed on Imaris version 7.6 (Bitplane) and ImageJ (National Institutes of Health). Images from the videos with corresponding time points were plotted in Illustrator CS6 (Adobe).
5
Multimodal Imaging of Cell Morphogenesis
6
Nanofluidic Visualization of DNA Chromatin
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Nanofluidic devices were fabricated in PDMS elastomer as previously described27 (link). The device consisted of an array of 90 μm long, rectangular channels with a cross-section of 50 × 70 nm2 (60-nm channel system) or 120 × 130 nm2 (125-nm system) and uncertainty in width and depth of ± 5 nm. Before fluorescence imaging, DNA was stained with YOYO-1 (Invitrogen, Carlsbad, CA) at a ratio of one dye molecule for each four base pairs. Then, the stained chromatin was driven into the nanochannels by electrophoresis. The fibers were visualized with a Nikon Eclipse Ti inverted fluorescence microscope equipped with a diode laser, 200 mW/488 nm (Omicron, Germany), filter set, and a 100× oil immersion objective (numerical aperture 1.49). Movie clips at a rate of 2.5 frames/s were recorded with an electron-multiplying charged coupled device camera (Andor iXon X3). The image pixel size of 0.16 × 0.16 μm2 was calibrated with the help of a metric ruler.
7
DNA Molecule Visualization in Nanochannels
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The pre-incubated and stained DNA molecules were loaded into one of the two reservoirs connected to the array of nanochannels. The DNA molecules were subsequently driven into the channels by electrophoresis. For this purpose, two platinum electrodes were immersed in the reservoirs and connected to a power supply with a relatively low voltage in the range 0.1–10 V (Keithley, Cleveland, OH, USA). Once the DNA molecules were localized inside the nanochannels, the electric field was switched off and the molecules were allowed to relax to their equilibrium state for at least 60 s. The stained DNA molecules were visualized with a Nikon Eclipse Ti inverted fluorescence microscope equipped with a 200 W metal halide lamp, a filter set, and a 100× oil immersion objective. A UV light shutter controlled the exposure time. Images were collected with an electron multiplying charge coupled device (EMCCD) camera (iXon X3, Andor Technology, Belfast, UK) and the extension of the DNA molecules inside the channels was measured with imageJ software (http://rsb.info.nih.gov/ij/ ). For intensity threshold, we have used two times the signal to background noise ratio.
8
High-Resolution Imaging Techniques for Cellular Investigations
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Imaging experiments (Figs. 3 C , 4 D , 5 A ; S1, A and F–I ; S2 D , and S3, D and F ) were performed with an inverted widefield microscope (Ti Eclipse; Nikon). A 100× Plan-Apochromat (NA 1.4) objective lens (Nikon) was used. Images were captured with a z-step size of 300 nm using a scientific complementary metal-oxide semiconductor camera (Zyla; Andor Technology).
Super-resolution localization experiments of SF–pcMT interactions (Figs. S1 C and S3 L ) and Group 2 SF proteins (Fig. 4 C ) were performed via SIM with the Nikon 3D SIM system (Ti 2 Eclipse). A 100× total internal reflection fluorescence objective (NA 1.45) was used. Images were captured with a complementary metal-oxide semiconductor camera (Orca-Flash 4.0; Hamamatsu) with a z-step size of 300 nm. Raw SIM images were reconstructed by the image stack reconstruction algorithm (Nikon Elements).
Confocal microscopy was performed using an inverted microscope (Ti Eclipse) with a 100× Plan-Apochromat (NA 1.43) objective lens (Nikon) and a swept field confocal scan head with the 35-µm slit mode (Prairie Technologies). Images were captured with a charge-coupled device camera (iXon X3; Andor Technology). Confocal images were also acquired with the A1 confocal laser microscope (Nikon). All images were acquired with Nikon Elements with a z-step size of 300 nm at room temperature (Figs. 1 , 2 , S1 E , and S2 A ).
Super-resolution localization experiments of SF–pcMT interactions (
Confocal microscopy was performed using an inverted microscope (Ti Eclipse) with a 100× Plan-Apochromat (NA 1.43) objective lens (Nikon) and a swept field confocal scan head with the 35-µm slit mode (Prairie Technologies). Images were captured with a charge-coupled device camera (iXon X3; Andor Technology). Confocal images were also acquired with the A1 confocal laser microscope (Nikon). All images were acquired with Nikon Elements with a z-step size of 300 nm at room temperature (
9
Immobilizing Adult Hermaphrodites for Live Imaging
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We improved a method to immobilize adult hermaphrodites for live imaging. Adult hermaphrodites at 24–36 h after the L4 stage were incubated with levamisole (0.5–1.5 mM) in M9 buffer for 10 min on a 10% agarose pad surrounded by a 0.5 mm thick silicone rubber frame. After incubation, a cover glass was placed over the worms. Samples were observed using an IX73 (Olympus) equipped with a 100 × 1.35 NA UPlanSApo, 60 × 1.30 NA silicone objective lens, or a 60 × 1.40 NA UplanSApo oil-objective lens, an EMCCD camera iXon X3 (Andor Technology, Belfast, Northern Ireland), and a confocal scanner unit CSU22 (Yokogawa, Tokyo, Japan). Images were acquired every 15 or 20 s at 1–1.5 μm intervals in the Z direction, and the maximum intensity projection was created using Micro-Manager software59 (link) or Andor iQ (Oxford Instruments).
10
Spinning-Disk Confocal Microscopy for Cell Imaging
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Imaging experiments were performed using an inverted microscope (Nikon Ti Eclipse) with a 60× Plan-Apo (NA 1.40) objective lens (Nikon) and a spinning-disk module (CSU-X1; Yokogawa). Images were acquired at room temperature. They were captured with a charge-coupled-device camera (iXon X3; Andor Technology) and Slidebook imaging software (3i—Intelligent Imaging Innovations). Image voxel size was 180 nm × 180 nm × 200 nm. To ensure accurate representation of BB organization and cell volume, care was taken to ensure that the entire cell volume was captured. Image acquisition conditions were kept constant across each of three biological replicates.
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