Cells were plated on poly-d -lysine–coated coverslips and transfected with Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer's recommendation. Cells were fixed in 4% formaldehyde (8 min) and permeabilized with 0.1% Triton X-100 in PBS with 5% BSA (10 min). After blocking with 5% BSA in PBS, primary antibodies were incubated overnight at 4 °C and stained with secondary antibodies for 1 h. Coverslips were mounted with SlowFade reagent (Life Technologies). Samples were imaged on a confocal microscope (A1R+, Nikon) equipped with GaAsP detectors and an oil objective (60×/NA1.40; Plan Apo) or on a STORM/TIRF microscope (Nikon) equipped with a laser (MLC400B, Agilent) charge-coupled device camera (iXon Ultra, Andor) and an oil objective (100×/NA1.49; ApoTIRF). The microscopes were controlled by Nikon Elements software. Image acquisition was performed at ambient temperature, and images were analyzed with ImageJ (National Institutes of Health) or Nikon Elements software.
Ixon ultra
Manufactured by Oxford Instruments
Sourced in United Kingdom
The IXon Ultra is a high-performance EMCCD camera designed for low-light imaging applications. It features a back-illuminated sensor with high quantum efficiency and low-noise performance, enabling the detection of single photon events. The camera is capable of high-speed readout and high-resolution imaging, making it suitable for a variety of scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (6 mentions)
Eclipse ti, by Nikon (5 mentions)
Fpl 1055 t, by Thorlabs (3 mentions)
Csu x, by Oxford Instruments (3 mentions)
Ichrome mle, by Toptica (3 mentions)
Du897 ex, by Oxford Instruments (2 mentions)
Ixon 3 860, by Oxford Instruments (2 mentions)
Uplansapo 100 xo, by Olympus (2 mentions)
Inug2 onics, by Tokai Hit (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Stage top incubator, by Okolab (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Uplansapo, by Olympus (2 mentions)
Fv1000 microscope, by Olympus (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Mlc400b, by Agilent Technologies (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Plan apo, by Nikon (1 mentions)
Elements software, by Nikon (1 mentions)
37 protocols using ixon ultra
1
Fluorescent Labeling of Cellular Proteins
2
In Vivo Fluorescence Imaging of MNPs
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The DCs labeled with ICGylated MNPs (2 × 106) were injected into the mouse footpads, and the imaging areas were treated with a depilatory cream. The mice were placed in an in-house-prepared high-quality in vivo imaging system connected to an EMCCD camera (DU897-EX, iXon Ultra; Andor Technology). Fluorescence images of the mice were acquired using an 808-nm (0–15 W) diode laser (OCLA, Gyeonggi-do, Korea) as an excitation light source and an emission filter (840 ± 12 nm, Semrock).
3
Organotypic PCLS Bioluminescence Imaging
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Organotypic PCLS were prepared as described before (2 (link)). Bioluminescence images were obtained using a 2.5× objective (Zeiss) and captured using a cooled Andor iXon Ultra camera over a 30-min integration period.
4
Confocal and TIRF Imaging of Live Cell Dynamics
5
High-speed Cardiac Myocyte Imaging
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The optical system used was similar to that used in our previous work, with some modifications17 (link),18 (link). Briefly, an inverted microscope (IX-70, Olympus) was equipped with a high-sensitivity camera (iXon 3 860, iXon Ultra, Andor Technology) and an oil immersion objective (UPlanSapo 100 XO, Olympus). The vibration noise of the microscope was reduced by making the microscope stage rigid and optimizing the performance of a vibration-free table. The size of a pixel on the camera corresponded to 150 nm in the sample. The frame rate was adjusted to 500 fps for high-temporal-resolution analysis. The temperatures of the glass slides seeded with cardiomyocytes were controlled to 37.0 ± 0.2 °C by a thermostatically controlled incubator on the sample stage (INUG 2—ONICS, Tokai Hit Co.). A 1550 nm laser (FPL 1055 T, Thorlabs) was used as a heat source to enable rapid alteration of temperature in the vicinity of the cardiac myocytes being monitored. A 488 nm laser (FITEL HPU50211 (Blue), Furukawa Electric) was used as an excitation light source for fluorescence observation of AcGFP-actinin.
6
Microtubule Sliding Motility Assay with OsDLK Motors
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Microtubules were polymerized as described before using DyLight594-labeled or a mixture of Cy5-labeled and digoxygenin-labeled (1:5) tubulin. For the microtubule sliding motility assay, microtubules co-labeled with digoxygenin and Cy5 were immobilized to the glass surface via digoxygenin antibodies (Roche). After blocking with 1% Pluronic F127, OsDLK motors were added to the microtubules in absence of ATP. Subsequently, microtubules labeled with DyLight594 in imaging solution were allowed to bind to the motors and transport was monitored in presence of 2 mM ATP. Fluorescently labeled microtubules were visualized using epi-illumination on an inverted fluorescence microscope (Ti-E, Nikon) equipped with an EMCCD camera (iXon Ultra, Andor). Positions of microtubules were obtained using FIESTA tracking software as described before63 (link). The mean velocity was determined by fitting the velocity histograms to Gaussian functions using MatLab (Mathworks).
7
Single-molecule imaging of Myo10 dynamics
8
Multimodal Microscopy Imaging Techniques
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The transmitted light and fluorescence microscopy imaging were performed using an inverted microscope (Eclipse-Ti; Nikon, Japan) configured with 20x and 40x dry objective lenses (Plan Apo 20x/0.75NA and 40x/0.95NA, respectively; Nikon, Japan). The transmitted light microscopy was conducted using a differential interference (DIC) contrast setup (Nikon, Japan) configured with white-light light-emitting-diode (LED) illumination (pE-100; CoolLED, UK). For the fluorescence microscopy, the sample was illuminated using colored LED light sources (pE-4000; CoolLED, UK). TRI, RFP, and Cy5 filters (Nikon, Japan) were used, depending on the fluorescence label. The microscopic images were recorded using an electron-multiplying charge-coupled-device camera (iXon Ultra; Andor, UK). A focus stabilization system (PFS; Nikon, Japan) was used for all imaging experiments. All the data were acquired by focusing fiducial markers immediately above the coverslip. The microscope system was controlled using MetaMorph software (Molecular Device, USA).
9
Dual-color Fluorescence Microscopy Imaging
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We performed cell imaging with an inverted microscope (Ti-2, Nikon) equipped with a confocal unit (Dragonfly 200, Andor Technology), a microscope objective (CFI Plan Apochromat λ 60 × Oil; numerical aperture, 1.40; Nikon), and a stage-top incubator with 5% CO2 supply (STXG-WSKMX, Tokai Hit). We used a 445 nm laser for excitation and collected the fluorescence emission through bandpass filters (ET480/40 nm and ET540/30 nm for mT and mV, respectively; Chroma). We captured the fluorescence images with an EMCCD camera (iXon Ultra, Andor Technology). The exposure time was 500 ms, and the binning size was 2 × 2 pixels. We measured fluorescence ratios of mV/mT cell by cell to calculate the average values and the standard deviation.
10
Live-Cell Imaging with Confocal and TIRF Microscopy
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All live cells were maintained at 37°C, 5% CO2 with a stage top incubator (Okolab) during imaging. For confocal microscopy, cells were imaged with a spinning disk confocal microscope (Eclipse Ti, Nikon) with a spinning disk (Yokogawa CSU-X, Andor), CMOS camera (Zyla, Andor), and either a 20x objective (Plano Fluor, 0.45NA, Nikonor a 60x objective (Apo TIRF, 1.49NA, oil, Nikon). For total internal reflection fluorescence (TIRF) microscopy, cells were imaged with TIRF microscope (Eclipse Ti, Nikon), 60x objective (Apo TIRF, 1.49NA, oil, Nikon) and EMCCD camera (iXON Ultra, Andor). Both microscopes were controlled with Micro-Manager. Images were analyzed and prepared using FIJI (Schindelin et al., 2012 (link)).
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