Images of cultures were taken with a Canon EOS 250D camera (Canon, Tokio, Japan) equipped with a Canon Compact-Macro EF 50 mm lens (Canon, Tokio, Japan), after 2 and 3 days of growth. For single cell imaging, cells were diluted to OD730 of 0.0175, 100 µL of each culture was transferred to a 96-well plate, and images were taken with a Nikon Eclipse Ti2-E-microscope (Nikon, Tokio, Japan) and Nikon DS-Fi3-camera (Nikon, Tokio, Japan).
Eclipse ti2 e microscope
Manufactured by Nikon
Sourced in Japan, United States
The Eclipse Ti2-E microscope is a high-performance inverted research microscope designed for advanced life science applications. It features a motorized, modular design that allows for versatile configuration and customization to meet the specific needs of researchers. The core function of the Eclipse Ti2-E is to provide high-quality imaging and analysis capabilities for a wide range of specimens and applications.
Automatically generated - may contain errors
Lab products found in correlation
Spectra x, by Lumencor (4 mentions)
Ds qi2 cmos camera, by Nikon (3 mentions)
Ixon ultra 897 emccd camera, by Oxford Instruments (3 mentions)
Sola se 2, by Lumencor (2 mentions)
Orca flash 4.0 scmos camera, by Hamamatsu Photonics (2 mentions)
Nis elements ar software, by Nikon (2 mentions)
A1r hd, by Nikon (2 mentions)
Hoechst dye, by Merck Group (2 mentions)
Nis elements ar, by Nikon (2 mentions)
Nis elements software, by Nikon (2 mentions)
Eos 250d camera, by Canon (1 mentions)
Ds fi3 camera, by Nikon (1 mentions)
Prime bsi scmos camera, by Teledyne (1 mentions)
Csu w1 spinning disk confocal unit, by Yokogawa (1 mentions)
Andor dragonfly 200, by Oxford Instruments (1 mentions)
Perfect focus system, by Nikon (1 mentions)
Nis elements acquisition software, by Nikon (1 mentions)
Ds qi2 camera, by Nikon (1 mentions)
Orca fusion digital cmos camera, by Hamamatsu Photonics (1 mentions)
Ar9961, by Leica (1 mentions)
45 protocols using eclipse ti2 e microscope
1
Imaging of Microbial Cell Cultures
2
Immunohistochemical Analysis of Murine Tissues
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Five-week-old wild-type BALB/c mice were maintained in the Laboratory Animal Center of Sun Yat-sen University. All animal studies were approved by the Institutional Animal Care and Use Committees of the Sun Yat-sen University (approval number: SYSU-IACUC-2020-B0778). Mice were sacrificed, and the trachea, lung, and liver were harvested for immunohistochemical analysis according to standard procedures [37 (link)]. The images from tissue sections were obtained and analysed using a Nikon Eclipse Ti2-E microscope (Nikon).
3
Quantifying MVB Fusion at Plasma Membrane
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The analysis of MVB fusion events at the plasma membrane was performed as in Verweij et al. with few modifications [34 (link)]. PC-3 cells grown in MatTek 3.5 cm dishes were transfected with SNAP29-5 and non-targeting siRNA as described in the siRNA transfection section. The medium was replaced 12 h after transfection to remove the transfection reagent and then cells were transfected with 1 μg CD63-pHluorin plasmid using Fugene 6 with 3:1 ratio of reagent to DNA. Live-cell imaging was performed 48 h after CD63-pHluorin transfection on a Nikon ECLIPSE Ti2-E microscope (Nikon Corp, Tokyo, Japan) using a CFI Plan Apo λ 100× (NA 1.54 Oil) objective, a CSU-W1 spinning disk confocal unit (50 µm pinhole size, Yokogawa Electric Corp, Tokyo, Japan), a Prime BSI sCMOS camera (Teledyne Photometrics, Tucson, AZ, US), 405 nm, 488 nm, 561 nm and 638 nm lasers, BrightLine bandpass filters (447/60, 525/50, 600/52, and 708/75), and NIS-Elements AR 5.30 software. Environmental control was provided by a stage-top incubator with temperature control, digital CO2 control, and active humidification (Okolab). Images of at least 5 random fields of view per condition were captured. Images were acquired at 1 Hz focusing at or near the plasma membrane. Image analysis was performed using the ImageJ2/Fiji plugin ExoJ [47 ]. On average, ≥ 10 cells were imaged per condition in ≥ 6 different fields of view.
4
Large-Scale 3D Tissue Imaging
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Tissue sections were imaged using an inverted Nikon Eclipse Ti2-E microscope (Nikon Instruments, Melville, NY, USA) attached to an Andor Dragonfly 200 spinning disk unit (Oxford Instruments, Abingdon, UK). For all the experiments, a Plan Apochromatic 10x with a numerical aperture (NA) of 0.45 was used. A high-precision motorized stage was used to collect the large-scale 3D mosaics of each tissue section. The tissue sections were imaged on a high-resolution scientific complementary metal oxide semiconductor (sCMOS) camera (Zyla 4.2, 2.0 Andor, Oxford Instruments Company). The fusion (Andor, Oxford Instruments Company) and Image J/Fiji (v1.51s, Wayne Rasband, NIH, Bethesda, MD, USA) software were used for the acquisition and image processing.
5
Widefield Epifluorescence Imaging Setup
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Widefield epifluorescence imaging was performed on an inverted Nikon Eclipse Ti2-E microscope (Nikon Instruments), equipped with XY-motorized stage, Perfect Focus System, and an oil-immersion objective (Apo 60×, NA 1.4, oil). Setup was controlled by NIS-Elements AR software (Nikon Instruments). Fluorescent light was filtered through 488 (AHF; EX 482/18; DM R488; BA 525/45), 561 (AHF; EX 561/14; DM R561; BA 609/54), and Cy5 (AHF; EX 628/40; DM660; BA 692/40) filter cubes. A fluorescent lamp (Lumencor Sola SE II) was used as a light source and emitted light was imaged with ORCA-Flash 4.0 sCMOS camera (Hamamatsu Photonics). Images were acquired at 16-bit depth, 1024 × 1024 pixels, and pixel size 0.27 µm.
6
Automated Cell Migration Monitoring
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For measurement of cell migration into a wound region, the IncuCyte® 96-well scratch wound cell migration and invasion system (Sartorius Austria GmbH, Vienna, Austria) was used. Briefly, the following cell amounts were seeded per well of the IncuCyte® Imagelock 96-well Plate: COS3600—3 × 104; COS3600B—1.5 × 104; COS4074—4.5 × 104; COS4288—2 × 104; D-17—1 × 104. A wound (scratch) was created using the IncuCyte® WoundMaker device according to the manufacturer’s recommendations. Wound closure was monitored using a Nikon ECLIPSE Ti2-e microscope equipped with a Nikon DS-Qi2 Camera (Nikon GmbH, Vienna, Austria). Brightfield pictures were taken at 10× magnification (pixel size (x/y): 0.72 µm) using a 3 h time series interval. The acquisition pipeline was scripted with the JOBS module of the NIS Elements acquisition software (Nikon GmbH). Based on automated well recognition, 2 × 2 stitched images (10% blending) were acquired at the well center for each time point. Images were analyzed using Fiji software [29 (link)]. Images were processed with a Gaussian filter (s = 2), followed by a Top Hat filter (r = 25). Subsequently, the wound area was measured based on threshold segmentation. Data represent the mean from six replicates.
7
Wound Healing Assay for Cell Migration
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Culture‐Insert (Ibidi) was used to perform wound healing assay to measure cell migration according to the manufacturer's instructions. Briefly, NPC cells were seeded onto 24‐well plates to create a confluent monolayer and Culture‐Insert was inserted to form a “scratch” simultaneously. After culturing for 12 h, Culture‐Insert was removed to form scratches and the first image of the scratch was acquired with a 20 × objective using a Nikon Eclipse Ti2‐E microscope (Nikon). The second image was acquired after H‐EVs (5 μg/ml) or L‐EVs (5 μg/ml) treatment for 24 h.
8
Pollination Dynamics in Arabidopsis
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Flowers that two days after emasculation from nerd1-2 plants were cross-pollinated with wild-type Col-0. The pollinated pistils were then collected at 36 h after hand pollination and fixed immediately in ethanol-acetic acid (3:1 v/v) overnight. Pistils were rehydrated in 70%, 50%, 30% ethanol for 5 min each at room temperature. After clearing in 5 N NaOH at 60°C for 5 min and washing 2 × in 0.1M phosphate buffer (pH-8), they were stained using aniline blue (0.1% aniline blue in K3PO4) for 15mins. Five pistils per sample were analyzed and images were captured with a Nikon Eclipse Ti2-E microscope (Nikon Instruments Inc, Melville, NY).
9
Widefield Epifluorescence and 3D dSTORM Imaging
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Widefield epifluorescence and 3D dSTORM imaging were performed on an N-STORM 4.0 microscope from Nikon Instruments. More specifically, this is an inverted Nikon Eclipse Ti2-E microscope (Nikon Instruments), equipped with XY-motorized stage, Perfect Focus System, an oil-immersion objective (HP Apo TIRF 100×H, NA 1.49, Oil) and N-STORM module. Setup was controlled by NIS-Elements AR software (Nikon Instruments). Fluorescent light was filtered through the following filter cubes: 488 (AHF; EX 482/18; DM R488; BA 525/45), 561 (AHF; EX 561/14; DM R561; BA 609/54), Cy5 (AHF; EX 628/40; DM660; BA 692/40) and Nikon Normal STORM cube (T660lpxr, ET705/72 m). Filtered emitted light was imaged with ORCA-Flash 4.0 sCMOS camera (Hamamatsu Photonics). For epifluorescent widefield imaging, fluorescent lamp (Lumencor Sola SE II) was used as a light source. For 3D dSTORM imaging, a 647 nm laser (LU-NV Series Laser Unit) was used and a cylindrical lens was introduced in the light path11 (link).
10
Quantitative Fluorescent Microscopy Protocol
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Fluorescent microscopy was performed at the Nikon Imaging Core (UCSD) using a Nikon Eclipse Ti2-E microscope with Plan Apo objectives. Samples were excited by the Lumencor SpectraX and acquired with a DS-Qi2 CMOS camera using NIS-Elements software, or with a laser scanning confocal (A1R HD, Nikon), acquired with an iXon Ultra 897 EMCCD camera (Andor). All AVPV slides were imaged at the same time and under the same conditions. All ARC and thalamic reticular nucleus slides were imaged at the same time and under the same conditions. The number of Kiss1 cells that colocalized with Six3 or atypical signal was determined manually, using FIJI Cell Counter tool. NIS-Elements: General Analysis software was used to objectively quantify the intensity of Kiss1, cFos, and Six3 signals. A signal threshold of 2.5 standard deviations above background was used for defining positive signal of each gene. cFos and Six3 signals were quantified only in Kiss1-positive cells.
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