All epifluorescent images of fixed specimens were acquired with a fluorescence microscope (Eclipse 80i; Nikon) using a 20×/0.75 NA or 60×/1.4 NA oil immersion objective lens fitted with a digital camera (DS-2; Nikon) and NIS-Elements BR 3.0 software (Nikon). Images were adjusted for brightness and contrast using ImageJ. Images from endothelial repulsion assays were acquired with an inverted florescent microscope (Eclipse TE2000-S; Nikon) using a 4×/0.2 NA objective lens, a digital camera (DS-5M; Nikon), and NIS-Elements BR 3.0 software (Nikon). Images were adjusted for brightness and contrast using ImageJ. Time-lapse videos were recorded at 37°C using an incubator live cell imaging microscope (VivaView FL LCV-110; Olympus) with a UPlan-SApochromat 40×/0.95 NA, WD 0.18 mm objective lens (Olympus) and a camera (Orca R2 CCD; Hamamatsu Photonics). Videos were acquired with MetaMorph for Olympus VivaView FL LCV-110 software, and separated images were adjusted for brightness and contrast using ImageJ.
Orca r2 ccd
Manufactured by Hamamatsu Photonics
Sourced in Japan, United States, Italy
The Orca R2 CCD is a high-performance, cooled charge-coupled device (CCD) camera from Hamamatsu Photonics. It is designed for scientific and industrial applications that require high-quality image capture and low-noise performance.
Automatically generated - may contain errors
Lab products found in correlation
Ds 5m, by Nikon (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Eclipse te2000 s, by Nikon (1 mentions)
Nis elements br 3, by Nikon (1 mentions)
Orca flash 4.0 cmos, by Hamamatsu Photonics (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Nis elements ar software, by Nikon (1 mentions)
Uv 2e c filter, by Nikon (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Nikon (1 mentions)
C10910 01, by Hamamatsu Photonics (1 mentions)
Ti wide field microscope, by Nikon (1 mentions)
Incubating chamber, by Okolab (1 mentions)
9 protocols using orca r2 ccd
1
Fluorescence Microscopy Imaging Protocol
2
Fluorescence Imaging of Cellular Structures
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It was performed with a Nikon Eclipse 90i microscope, equipped with CFI PLAN APO VC (NA 1.40) oil immersion objective and a Hamamatsu ORCA-R2 CCD camera. A red filter with excitation 543/22 and emission 593/40 was used. Differential interference contrast (DIC) images were also captured.
3
Imaging Techniques for Hermaphrodite Worms
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Adult hermaphrodites were anesthetized with tricaine/tetramisole in PBS and immobilized between a coverslip and agarose pad on a slide. Imaging for Figs. 1 , 2 , 3 , 4A , and 5G was done using MicroManager with a microscope (IX81; Olympus) equipped with an oil objective (60× PlanApo 1.42), a disk-scanning unit (Olympus), and a camera (ORCA Flash 4.0 CMOS; Hamamatsu Photonics). Imaging for Figs. 4 (C and D) and 5 (A–F, J, and K) were captured using Velocity on a Cetus Ultraview Spinning Disk Confocal microscope (PerkinElmer) equipped with a camera (Orca R2 CCD; Hamamatsu Photonics) and an oil objective (100× 1.35; Olympus). Fig. 2 (A–C) was deconvolved using Huygens Professional X11 (SVI). Fig. 5 G , Fig. S1, and Videos 1–5 were processed using ImageJ (National Institutes of Health) Rolling Ball Background Subtraction.
4
Imaging of Protein Localization in Cells
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MLO-Y4 cells were transfected by electroporation, in triplicate with plasmids encoding either Src-Venus, MBD2-Venus or Pyk2-Venus and transferred to individual wells of 6-well culture plates. Non-transfected cells were grown in a separate well and the plates were placed in an incubator (37°C and 5% CO2-Air 95%) prior to imaging and recovery of lysates for Western blotting the following day. The cells were imaged for brightfield and Venus FP fluorescence using an Olympus IX71 microscope equipped with a 10-X air objective. The fluorescence images were acquired with a Semrock YFP-2427A filter and captured with a Hamamatsu Orca R2 CCD (charge-coupled device) camera using Micromanager and ImageJ software. For overlay, the fluorescence image was converted to indexed image and a lookup-table from red to white was applied using Vistavision software (ISS Inc., Champagne, IL). This image was then merged with the corresponding bright-field image.
5
Microscopic Imaging of Bacterial Cells
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Bacterial cells were observed with a Nikon Eclipse 90i microscope, equipped with a CFI PLAN APO VC 100x (NA 1.40) oil immersion objective and a Hamamatsu ORCA-R2 CCD camera. For mCherry fluorescence, a 543/22 nm excitation and 593/40 nm emission filter and 200 ms exposures were used. Differential interference contrast (DIC) shots (100 ms) were also captured. Images were analyzed using the NIS-Elements AR software (Nikon). Bacterial culture aliquots were fixed in formaldehyde and mounted on poly-L -lysine coated slides, as described in Fernández-Tresguerres et al. (2010) (link).
6
Fluorescence Microscopy of Fixed Cells
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Cells were fixed with paraformaldeyde 2% for 30 min at room temperature; after fixation, cells were washed once with PBS and stained with Hoechst 33342 (1 μg/ml in PBS, Invitrogen-Life Technologies) for 30 min. Fluorescence and phase contrast images were acquired by using an Eclipse Ti inverted microscope (Nikon, Tokyo, Japan) equipped with a × 20 air Nikon objective, N.A. 0.75, ApoFluor and a CCD ORCA R2 (Hamamatsu, Shizuoka, Japan). Hoechst 33342 was excited by a mercury arc lamp using a 450/50 nm band-pass filter, and emission was collected using an UV-2E/C filter (Nikon). Cell death was qualitatively assessed on the basis of cellular and nuclear morphology evaluating the presence of condensed or fragmented nuclei.
7
Ultrafast Fluorescence Characterization
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The fluorescence measurements were carried out at 298 K on sample solutions placed in 10 mm-thick quartz cuvette. A frequency-tripled (λexc = 355 nm, beam waist = 4 mm) Q-switch Nd:YAG laser (Brio, Quantel) with 4 ns pulse duration and 20 Hz repetition rate was used to investigate the steady-state fluorescence, whose spectra were recorded with a fiber-spectrometer (USB200+UV-Vis, Ocean Optics, 600 lines per mm, 25 μm slit) in the range 320–750 nm. Time-resolved fluorescence spectroscopy measurements have been carried out by irradiating the samples with 100 fs laser pulses (Coherent Libra + OPerA Solo, λexc = 355 nm, beam waist = 4 mm, P = 20 μJ, rep. rate = 1k Hz) and collecting the emission with a spectrograph (Acton SP2358i, 0.3 m focal length, 150 lines per mm, 20 μm slit) equipped with a streak camera (Hamamatsu, C10910-01 + Slow Single Sweep module M10913-01 + CCD ORCA-R2) in the spectral range 470–570 nm and temporal window of 100 ns.
8
Fibroblast migration analysis protocol
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WT and HOM fibroblasts were seeded at a density of 1.5 × 104 cells/cm2, and, the day after (24 ± 1 h), live-imaged for 24 h, every 15 min, with an inverted Nikon-Ti wide-field microscope (Nikon, Tokyo, Japan) equipped with perfect focus system and cell incubator (Okolab, Naples, Italy), with an air 20× (NA 0.45, Plan-Fluor, Irving, TX, USA) objective, and a CCD ORCA R2 (Hamamatsu, Iwata City, Japan).
Cell movies were analysed with the ImageJ manual tracking plugin MTrack as stated by Tonazzini and colleagues [41 (link)]. We measured (i) the average speed (V, the mean displacement achieved in 1 h; in μm/h), (ii) the total displacement (the total distance covered by each cell from t = 0 to t = 24 h; in μm).
We analysed at least 10 cells for each sample, and we performed n = 4 independent experiments for each condition.
Cell movies were analysed with the ImageJ manual tracking plugin MTrack as stated by Tonazzini and colleagues [41 (link)]. We measured (i) the average speed (V, the mean displacement achieved in 1 h; in μm/h), (ii) the total displacement (the total distance covered by each cell from t = 0 to t = 24 h; in μm).
We analysed at least 10 cells for each sample, and we performed n = 4 independent experiments for each condition.
9
Proliferation of RT4-SCs on Chitosan Membranes
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RT4-SCs cells were seeded on chitosan membranes at a concentration of 10,000 cells/cm2 (t = 0) and cultured up to 72 h. The cultures were imaged at 24, 48 and 72 h using a Nikon Eclipse-Ti inverted wide-field microscope (Nikon, Tokyo, Japan) equipped with a 20× air Nikon objective (NA 0.45, Plan-Fluor), an incubating chamber (Okolab, Naples, Italy) and a CCD ORCA R2 (Hamamatsu, Iwata City, Japan). In order to assess the proliferation rate on the chitosan membranes, the GFP-positive cells were quantified on different samples at 24 and 72 h using an ImageJ program (NIH, Bethesda, MD, USA), and their concentration was expressed as cells/mm2. At least 10 images/sample were analyzed. We performed n ≥ 3 independent experiments for each condition.
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