Orca flash4 cmos camera

Manufactured by Hamamatsu Photonics

The Orca Flash4 is a CMOS camera produced by Hamamatsu Photonics. It is designed to capture high-resolution images and videos. The camera features a large sensor size, high frame rate, and low noise performance.

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Lab products found in correlation

Ix70 microscope, by Yokogawa (2 mentions) Rat tail collagen 1, by BD (1 mentions) Axio observer inverted microscope, by Zeiss (1 mentions) Axio observer microscope, by Zeiss (1 mentions) Dmribe microscope, by Leica (1 mentions) Cascade 512 camera, by Teledyne (1 mentions) Eclipse ti inverted microscope, by Nikon (1 mentions)

5 protocols using orca flash4 cmos camera

Visualizing B. theta Uptake by BMDMs

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BMDM were plated on a 96 well glass bottom plate with #1.5 cover glass and a black frame (CellVis) at 1×10⁵ cells per well in I-10 media overnight. The cells were stained with 5 μM CellTracker™ Orange CMTMR Dye for 37 °C x 30 min then washed. Cells were washed and incubated in 150 μL I-10 media. 50 μL of a 1:31 dilution of single CPS-expressing B. theta strains that endogenously expressed GFP with or without 10 μg/mL of B. theta antibody was added to each well. B. theta strains expressing a single CPS and GFP were grown in a 2mL TYG culture at 37 °C overnight to mid log phase. Cultures were washed once and resuspended in PBS prior to adding to the assay. Four hours later, images were acquired in the same z-plane using an Olympus IX70 microscope with a 100x/1.4NA oil objective, a Yokogawa spinning-disk confocal scanning unit, and a Hamamatsu Orca Flash4 CMOS camera. Cells were maintained at 37 °C with 5.0% CO₂ in a Tokai Hit humidified chamber. Images were acquired with NIS-Elements AR software and the number of bacteria uptaken per BMDM was measured manually using Imaris software. A total of 150 individual cells were measured for each bacterial strain analyzed with 50 cells measured per experiment in 3 experiments.

Hsieh S., Porter N.T., Donermeyer D.L., Horvath S., Strout G., Saunders B.T., Zhang N., Zinselmeyer B., Martens E.C., Stappenbeck T.S, & Allen P.M. (2020). Polysaccharide Capsules Equip the Human Symbiont Bacteroides thetaiotaomicron to Modulate Immune Responses to Dominant Antigen in the Intestine. Journal of immunology (Baltimore, Md. : 1950), 204(4), 1035-1046.

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Imaging and Analyzing Macrophage Podosome Dynamics

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Macrophages isolated from naive LifeACT-RFP⁺-WT and LPL^−/− mice were placed on coverslips pre-treated with Rat Tail Collagen 1 (BD Biosciences, 47.3 μg/ml in acetic acid). Cells were serum-starved for 1 h and then re-exposed to serum immediately prior to imaging. Images were collected from the first cell in which podosomes appeared. Cells were imaged for 30 min, with two cells imaged consecutively after serum re-exposure. Images were acquired in the same z-plane every 15–20 s using an Olympus IX70 microscope with a 100×/1.4NA oil objective, a Yokogawa spinning-disk confocal scanning unit, and a Hamamatsu Orca Flash4 CMOS camera. Cells were maintained at 37 °C with 5.0% CO₂ in a Tokai Hit humidified chamber. The microscope was controlled and images were acquired with Micro-Manager 1.4.22 and ImageJ software. Images were converted to videos with ImageJ, using the Images to Stack function with the Time Stamper plugin.
Videos records of podosome turnover were assessed by a blinded observer, and individual podosomes were tracked manually using ImageJ and the MTrackJ plugin. Podosome duration, total podosome counts, and intensity over time were calculated (TimeSeriesAnalyzer plugin). Heat maps of intensity over time were generated from the raw data with a script in the open-source software R (www.r-project.org/).

Zhou J.Y., Szasz T.P., Stewart-Hutchinson P.J., Sivapalan J., Todd E.M., Deady L.E., Cooper J.A., Onken M.D, & Morley S.C. (2016). L-Plastin promotes podosome longevity and supports macrophage motility. Molecular immunology, 78, 79-88.

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Multimodal Optical Imaging Protocol

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Optical imaging was performed on a Zeiss Axio Observer inverted microscope. A mercury lamp provided the fluorescent light source for imaging. A QV2 multichannel imager (Photometrics) was attached via a side documentation port and used a sequence of beamsplitters to separate the emitted fluorescent light from the sample onto different quadrants on a Hamamatsu Orca Flash4 CMOS camera. The fluorescence and phosphorescence signals were filtered at 470 nm and 528 nm, respectively, with Δλ = 25 nm.

Samonina-Kosicka J., Weitzel D.H., Hofmann C.L., Hendargo H., Hanna G., Dewhirst M.W., Palmer G.M, & Fraser C.L. (2015). Luminescent Difluoroboron β-Diketonate PEG-PLA Oxygen Nanosensors for Tumor Imaging. Macromolecular rapid communications, 36(7), 694-699.

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Multimodal Fluorescence Imaging Microscopy

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Fluorescence optical imaging was performed on an inverted Zeiss Axio Observer microscope with a mercury lamp. A QV2 multichannel imager (Photometrics) employed a sequence of beamsplitters to separate the emitted fluorescent light from the sample onto different quadrants on a Hamamatsu Orca Flash4 CMOS camera. The fluorescence and phosphorescence signals were filtered at 505 nm and 565 nm, respectively, with Δλ = 25 nm.

DeRosa C.A., Samonina-Kosicka J., Fan Z., Hendargo H.C., Weitzel D.H., Palmer G.M, & Fraser C.L. (2015). Oxygen Sensing Difluoroboron Dinaphthoylmethane Polylactide. Macromolecules, 48(9), 2967-2977.

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Immunohistochemistry of Brain Samples

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Sampled brains were embedded in OCT (Tissue-Tek, Torrance, CA) and frozen in isopentane at -25°C. 20 μm sagittal sections were cut from frozen brains in a cryostat and fixed in 4% paraformaldehyde for 15 min at 21°C. Serial sections were permeabilized for 60 min in PBS containing 0.25% Triton X-100 and 5% newborn goat serum, prior to incubation with primary antibodies at the following dilutions: anti-GFAP (1:500), anti-PN capsular (1:300), and AlexaFluor 568-IB4 (1:100). Alex-conjugated secondary antibodies and Phalloidin were used at a 1:200 dilution. DAPI was used at a 0.1 mg/ml final concentration. Samples were mounted in DAKO fluorescence mounting medium (DAKO Corp.). Fixed samples were analyzed using Eclipse Ti inverted microscopes (Nikon) equipped with a 60 x objective, a CSUX1-A1 spinning disk confocal head (Yokogawa) and a Coolsnap HQ2 camera (Roper Scientific Instruments), or a CSU1-W1 confocal head (Yokogawa) and an ORCA Flash4 CMOS camera (Hamamatsu) controlled by the Metamorph 7.7 software. For live calcein assays and Ca²⁺ imaging, epifluorescence microscopy was performed using a DMRIBe microscope (LEICA microsystems) using 380 nm, 470 nm, or 546 nm LED source excitation, equipped with a Cascade 512 camera (Roper Scientific) driven by the Metamorph (7.7) software. Images were analyzed using the Metamorph software.

Bello C., Smail Y., Sainte-Rose V., Podglajen I., Gilbert A., Moreira V., Chrétien F., Cohen Salmon M, & Tran Van Nhieu G. (2020). Role of astroglial Connexin 43 in pneumolysin cytotoxicity and during pneumococcal meningitis. PLoS Pathogens, 16(12), e1009152.

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