All proteins/RNA (12 × UG repeats) samples were prepared in 20 mM Na2HPO4/NaH2PO4, pH 7.5, 75 mM NaCl, 2.5% glycerol, and 1 mM DTT. The 25-µL sample was plated on a 30-mm No. 1 round glass coverslip and mounted on an Observer D1 microscope with 100×/1.45 oil immersion objective (Zeiss). Protein droplets were viewed using HAL 100 halogen lamp, and images were captured with an OrcaD2 camera (Hamamatsu) using VisiView 4.0.0.13 software (Visitron Systems GmbH). For the time course experiment, full-length CIRBP droplet formation was induced by diluting CIRBP from a 10× buffer to a 1× buffer at the concentration of interest in the absence or presence of RNA. For CIRBPRGG, droplet formation was induced after addition of RNA.
Orcad2 camera
Manufactured by Hamamatsu Photonics
Sourced in Japan
The OrcaD2 camera is a high-performance scientific imaging camera designed for demanding applications. It features a large sensor with high-resolution and low-noise characteristics, enabling accurate and precise data capture. The camera's core function is to provide researchers and scientists with a reliable tool for capturing high-quality images and data for their scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Observer d1 microscope, by Zeiss (5 mentions)
Oil immersion objective, by Zeiss (4 mentions)
Visiview 4, by Visitron (4 mentions)
Eclipse ti, by Nikon (1 mentions)
Tetraspeck bead, by Thermo Fisher Scientific (1 mentions)
Elyra s 1, by Zeiss (1 mentions)
Ixon 885 emccd camera, by Oxford Instruments (1 mentions)
Ixon du897 ultra, by Oxford Instruments (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
Hcimage software, by Hamamatsu Photonics (1 mentions)
Dmi8 inverted microscope, by Hamamatsu Photonics (1 mentions)
Eclipse ni e microscope, by Nikon (1 mentions)
α bungarotoxin, by Bio-Techne (1 mentions)
Elements software, by Nikon (1 mentions)
10 protocols using orcad2 camera
1
Protein Droplet Formation Dynamics
2
Multimodal Microscopy Imaging Protocol
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Widefield and structured illumination images were acquired with a structured illumination microscope (Elyra S1 (Carl Zeiss, Jena, Germany) equipped with a 63x oil objective lens with a numerical aperture (NA) of 1.4 and an Andor iXon 885 EM-CCD camera). PALM imaging was performed using a Nikon Instruments Ti Eclipse inverted microscope with a 100x/1.49 NA TIRF objective (Apo) and a 488 nm laser (Agilent, MLC-MBP-ND laser launch) with an EM-CCD camera (Andor Technology, iXon DU897-Ultra). The microscope was equipped with a Perfect Focus Motor to minimize axial drift over the duration of imaging. paGFP was simultaneously activated and excited with the 488 nm laser at an intensity set to 1.45–1.9 mW (as measured at the optical fiber). Exposure time was set at 100 ms. Imaging was performed until paGFP was completely exhausted, typically after 20,000 frames. TetraSpeck beads (Life Technologies) were used as fiducial markers for drift-correction during image acquisition. TIRF images were acquired by a Nikon Ti Eclipse inverted microscope, equipped with a 100x/1.49 NA oil objective lens and a Hamamatsu Orca D2 camera, or Hamamatsu Orca Flash 4 Lite, for living cell imaging. Confocal images were acquired using the same microscope equipped with Nikon A1R using a 60x 1.4 NA oil objective. Co-localization analysis was carried out using the Fiji plugin JACoP [61 (link)].
3
Quantifying Tyrosine Hydroxylase Neurons with α-Synuclein Aggregates
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Manual cell counting operations were carried out with a Nikon Eclipse Ti-U fluorescence inverted microscope (Nikon France, Champigny sur Marne, France) equipped with a Hamamatsu’s ORCA-D2 camera and HCImage software (Hamamatsu Photonics, Massy, France). The number of tyrosine hydroxylase immunopositive (TH+) neurons/culture well was estimated by visually inspecting samples with a 10× objective over 10–15 visual fields that were randomly selected for each treatment condition. The number of TH+ somas containing αS aggregates (αSa) was estimated by visual examination of 10 visual fields that contained at least one TH+ neuron but were otherwise randomly chosen. The total number of Microtubule-Associated Protein-2 (MAP-2+) neurons and the number of MAP-2+ somas with αSa were estimated using 10 microphotographs of midbrain cultures that were acquired randomly with a 20X objective.
4
Microfluidic Chip Worm Imaging
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Microfluidic chips were made by polydimethylsiloxane (PDMS) replica molding from an SU-8 master mold fabricated by photolithography, as previously described45 . During imaging or genetic screening, age-synchronized worms were suspended in M9 buffer with 0.01% Triton X-100. A sealed plastic vial containing the worm suspension connected to the inlet of the microfluidic chip and to the pressure source was used to inject worms into the chip by pressure-driven flow. Imaging was performed at × 40 magnification in a compound microscope using an oil objective (numerical aperture=1.4) with a Hamamatsu Orca D2 camera for simultaneous imaging of the red and green channels.
5
FUS Protein Phase Separation Dynamics
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FUSRGG3PY and meFUSRGG3PY samples were prepared in 50 mM sodium succinate pH 6.0, 150 mM NaCl, 2 mM TCEP, 0.04% NaN3. The 30-μL sample was plated on a 30 mm No. 1 round glass coverslip and mounted on an Observer D1 microscope with 100×/1.45 oil immersion objective (Zeiss, White Plains, NY, USA). Protein droplets were viewed using a HAL100 halogen lamp, and images were captured with an OrcaD2 camera (Hamamatsu, Japan) using VisiView 4.0.0.13 software (Visitron Systems GmbH, Puchheim, Germany). The formation of droplets was induced by the addition of RNA/EGCG to all protein samples, and pictures were recorded for a duration of 1 h after the addition of RNA/EGCG.
6
Visualizing Droplet Formation of CIRBP-RGG
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CIRBP-RGG, pCIRBP-RGG and RNA (12 × UG repeats) samples were prepared in 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 2 mM TCEP, 0.04% NaN3.The 30-μL sample was plated on a 30-mm No. 1 round glass coverslip and mounted on an Observer D1 microscope with 100×/1.45 oil immersion objective (Zeiss). Protein droplets were viewed using HAL100 halogen lamp, and images were captured with an OrcaD2 camera (Hamamatsu) using VisiView 4.0.0.13 software (Visitron Systems GmbH). Droplet formation was induced by the addition of RNA for all proteins, and pictures were recorded for 30 min after addition of RNA.
7
Visualizing CIRBP and RNA Droplets
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CIRBP and RNA (12 × UG repeats) samples were prepared in 50 mM NaH2PO4/Na2HPO4, pH 6.5, 150 mM NaCl. Thirty microliter of sample was plated on a 30 mm No. 1 round glass coverslip and mounted on an Observer D1 microscope with 100×/1.45 oil immersion objective (Zeiss). Protein droplets were viewed using HAL 100 halogen lamp, and images were captured with an OrcaD2 camera (Hamamatsu) using VisiView 4.0.0.13 software (Visitron Systems GmbH). Droplet formation was induced by addition of RNA.
8
Bead Sorting by Fluorescence Microscopy
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In this study, beads are sorted based on their fluorescence intensity or pattern in both red and green channels. Simultaneous dual color fluorescence microscopy was performed using a LEICA DMi8 inverted microscope connected to a Hamamatsu Orca-D2 camera equipped with two charge-coupled devices (CCDs) enabling simultaneous microscopy in wavelengths of interest. The ChemMatrix beads used in this study tend to aggregate in solution, leading to clogging of the microfluidic platform and sorting errors (false positives/negatives). To prevent aggregation, beads were maintained in a diluted suspension in PBS buffer (150 beads per 80 ml) and gently stirred on an orbital shaker throughout the duration of the sorting cycle. On-chip valves were filled and degassed with a 50% glycerol solution with a similar refractive index as PDMS, which improves image quality in the vicinity of the valves. A custom-built pressure box equipped with pressure regulators was used to drive fluid flow in the tubing and device. Valve operations was controlled by a custom-developed MATLAB Graphical User Interface (GUI).
9
Calcium Imaging of Mechanosensitive Hair Cells
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For calcium imaging, measurements were made as previously described (Kindt et al., 2012 (link); Zhang et al., 2016 (link)). Briefly, larvae were anesthetized with 0.03% 3-amino benzoic acid ethylester (MESAB, Western Chemical, Ferndale, WA) in E3, and mounted with tungsten pins onto a Sylgard recording chamber. Larvae were then microinjected in the heart with 125 μM α-bungarotoxin (Tocris, Bristol, UK) to suppress muscle activity. After paralysis, calcium imaging was performed in extracellular solution in mM: 130 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2 and 10 HEPES, pH 7.3, 290 mOsm. A pressure clamp (HSPC-1, ALA Scientific, New York, NY) attached to a glass pipette (tip diameter ~30–50 μm) was filled with extracellular solution and used to mechanically stimulate HCs along the anterior-posterior axis of the fish. Calcium measurements were made on a Nikon Eclipse NiE microscope using a 60 × 1.0 NA CFI Fluor water-immersion objective and the following filter set: excitation: 480/30 and emission: 535/40. The microscope was equipped with an Orca D2 camera (Hamamatsu, Hamamatsu City, Japan), and images were acquired using Elements software (Nikon Instruments Inc., Melville, NY).
10
Phase Transition Dynamics of p53 Proteins
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All proteins (p531−94, p53TAD2, p53DBD, and p531−312) and PR25/GR25 were prepared in 20 mM Hepes pH 7.0, 50 mM NaCl, 2 mM TCEP, and 0.04% (w/v) NaN3. The 30 μL sample was plated on a 30 mm No. 1 round glass coverslip and mounted on an Observer D1 microscope with 100×/1.45 oil immersion objective (Zeiss, Oberkochen, Germany). Protein droplets were viewed using HAL 100 halogen lamp, and images were captured with an OrcaD2 camera (Hamamatsu, Shizuoka, Japan) using VisiView 4.0.0.13 software (Visitron Systems GmbH, Puchheim, Germany). Droplet formation was induced by the addition of PR25/GR25 in a fixed concentration of protein. Images were taken every 5 min until 30 min after the addition of peptides. Microscopy images were processed using Fiji/ImageJ 1.53a software (Bethesda, US), applying linear enhancement for brightness and contrast.
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