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Orca 2 er

Manufactured by Hamamatsu Photonics
Sourced in Japan, United Kingdom

The Orca II ER is a high-performance, back-illuminated CMOS camera designed for scientific and industrial applications. It features a large sensor size, high quantum efficiency, and low readout noise, making it suitable for a wide range of imaging tasks. The camera's core function is to capture high-quality images and data with high sensitivity and low noise levels.

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21 protocols using orca 2 er

1

Quantitative Bioluminescent Imaging

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C17.2 reporter cells were plated on 35mm glass-based dishes (Greiner-Bio One, UK) and were allowed to adhere before serum withdrawal for 3 hours and subsequent imaging in the presence of 10% serum and 1mM D-luciferin (Promega, UK). Plates were placed on an inverted Zeiss microscope stage and maintained at 37°C in 5% CO2. Luminescent images were obtained using a 10x 0.3NA air objective and collected with a cooled charge-coupled device camera (Orca II ER, Hamamatsu Photonics). A 30 minute exposure and 2x2 binning was used.
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2

Microscopy Imaging of Bacterial Cells

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C. crescentus cells were grown up to exponential phase (OD660nm < 0.3) and spotted on 0.3% agarose pads containing M2G medium, unless specified otherwise. Microscopy was performed on an Eclipse 80i microscope (Nikon, Tokyo, Japan) equipped with a phase-contrast objective Plan Apochromat 100×/1.40 NA (Nikon, Tokyo, Japan), an Orca-II-ER (Hamamatsu Photonics, Hamamatsu City, Japan) and an Andor iXon DU-897E camera (Andor Technology Ltd., Belfast, UK) with 2× optivar. Images were acquired every 2.5 min using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). For still images of E. coli strains, cells were grown at 30°C up to exponential phase (OD600nm<0.3) and spotted on 1% agarose pads. For microfluidic experiments, E. coli cells were loaded and grown for at least 5 generations in the microfluidic device prior to imaging. Microscopy was performed on an Eclipse Ti-E microscope (Nikon, Tokyo, Japan) equipped with Perfect Focus System (Nikon, Tokyo, Japan) and an Orca-R2 camera (Hamamatsu Photonics, Hamamatsu City, Japan) and a phase-contrast objective Plan Apochromat 100×/1.45 NA (Nikon, Tokyo, Japan). Time-lapse images were acquired every 5 sec using NIS-Element Ar software (Nikon Instruments INC., Melville, NY USA).
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3

Live Imaging of Microglia Cytoskeleton

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Homeostatic microglia cells were infected with EB3-EGFP lentivirus the day after plating for 24 h and live-cell imaging of EB3 comets was performed (2 s/frame for 3 min) in astrocytes conditioned medium/DMEM 2.5% FBS (1:1) using an epifluorescence microscope (Nikon Eclipse Ti) equipped with an Orca II ER charge-coupled device (CCD) camera (Hamamatsu, Hamamatsu, Japan) and a temperature-controlled (37°C) CO2 incubator using a 603/1.40NA objective. Kymographs were generated with ImageJ software.
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4

Immunofluorescence on Cryosections for Nerves

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Immunofluorescence on cryosections was performed as described (47 (link)) and examined with confocal TCS SP5 laser-scanning confocal (Leica) or Olympus BX (Olympus Optical) fluorescent microscope, and Zeiss Axiovert S100 TV2 with Hamamatsu OrcaII-ER. For immunohistochemistry, sciatic nerves were removed and rapidly snap-frozen in liquid nitrogen, either unfixed or previously fixed in buffered 4% PFA.
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5

Bioluminescence Monitoring of SCN Slices

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Bioluminescence signals from SCN cultures were recorded as described previously (Inagaki et al. 2007 (link)). The coronal SCN slices (100 µm thick) at the middle of the rostro–caudal axis were prepared from PER2∷LUC mice (Yoo et al. 2004 (link)) using a microslicer at ZT6–ZT10, and the bioluminescence signals from the slices were recorded by LumiCycle (Actimetrics). Single-cell analysis was performed using Cellgraph (ATTO) equipped with a highly sensitive cryogenic CCD camera (ORCA-II ER or ImagEM, Hamamatsu Photonics). Individual cellular rhythms were analyzed using Aquacosmos software (Hamamatsu Photonics), and distribution of the phases was analyzed by circular statistics software (Oriana, Kovach Computing Services). The bioluminescence signals were recorded after the slice was set on the apparatus (at time 0). In experiments using a drug, the medium was replaced with a medium containing KN92, KN93, or DMSO 96 h after the setting.
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6

Proliferation and Migration Assays for C2C12 Cells

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For the proliferation assay, C2C12 cells were plated at the density of 2500 cells/cm2. The day after, the transient transfection with siRNAs was performed as described above. Cells were counted every 9 or 15 h until 100% of confluence was reached. Doubling times were evaluated as follow: DT = (duration)Log(2))/((Log(final concentration) − Log(initial concentration)).
For the migration assay, C2C12 cells were transfected with siRNAs as described above. At the density of 60%, images were recorded every 5 min for 4 h, using Zeiss Axiovert S100 with Hamamatsu OrcaII-ER. Three fields with ×10 magnification were analyzed for each condition, and 11 mononucleated cells were tracked for each field. The mean of the total distance covered by each cell of the field were evaluated and converted from pixel to μm. The results are expressed as mean of three independent experiments, for a total of 99 cells per condition.
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7

Calcium Imaging and Luminescence of GH3/prolactin-luc Cells

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GH3/prolactin-luc cells were seeded in 35-mm glass coverslip-based dishes (IWAKI, Japan) 20 h prior to imaging. Luciferin (1 mM) was added at least 10 h before the start of the experiment, and the cells were transferred to the stage of a Zeiss Axiovert 200 equipped with an XL incubator (maintained at 37°C, 5% CO2, in humid conditions) maintained within a darkened room. Cells were loaded with Fluo-4 for 30 min and then time-series imaging was performed using a Fluar x20, 0.75 NA (Zeiss) air objective, with an argon-ion laser at 488 nm. Emitted light was captured through a 505–550 nm bandpass filter from a 545 nm dichroic mirror. Calcium recordings were captured every 1 s for at least 250 s unless stated otherwise. Data were captured using LSM510 software with consecutive autofocus. The microscope and all light emitting devices were then shut down and luminescence images were captured using a photon-counting charge coupled device camera (Orca II ER, Hamamatsu Photonics, UK). Sequential images, integrated over 30 min, were taken using 4 × 4 binning and acquired using Kinetic Imaging software AQM6 (Andor, UK). Bright field images were taken before and after luminescence imaging to allow localization of cells. In the relevant experiments, 0.5 µM BayK8644 was added to the dish at around 100 s during the calcium imaging period.
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8

Quantitative Time-Course Imaging of Competition

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For time-course imaging of competition experiments, cultures were set up as described above, with the addition of IPTG to the starting overnight culture and to the competition flasks to induce fluorescent reporter expression. At each time point, cells were harvested, diluted in one volume PYE and propidium iodide added to a final concentration of 15 µM. Cells were incubated in the dark for 15 min, spotted onto PYE 1.5% agarose pads and imaged immediately. Phase contrast and fluorescent images were taken on a Zeiss Axiovert 200M microscope with a 63× phase or αFluar 100×/1.45 oil immersion objective, using a digital camera (Orca-II ER; Hamamatsu Photonics) and Metamorph software (Universal Imaging, PA). The following emission/excitation filters were used: YFP, 500/25 and 535/30m; CFP, 436/25 and 480/40; mCherry, 560/40 and 630/75. Image analysis to quantify fluorescent signals for each cell was calculated using MicrobeJ (Ducret et al., 2016 (link)). Three independent replicates were analyzed, with a minimum of 250 cells per frame. Fold induction for the PcdzC-YFP transcriptional reporter was calculated relative to fluorescence density at OD600 = 0.025. Image overlays were generated using Fiji (Schindelin et al., 2012 (link)).
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9

Immunocytochemical Analysis of Cytoskeletal Proteins

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Briefly, for immunocytochemical analyses, cell cultures were rinsed in PBS and fixed for 15 min in 4% paraformaldehyde/PBS. Coverslips were then washed three times in PBS and incubated for 30 min in blocking solution (2% goat serum, 2% serum albumin, 0.1% Triton X-100 in PBS). Antibodies were diluted to the appropriate concentration in blocking solution. Coverslips were incubated for 60 min in the antibody solution. The samples were subsequently washed three times and incubated for 30 min with the appropriate fluorescence-conjugated secondary antibodies. Finally, coverslips were washed five times and mounted with Mowiol. Fluorescence images were obtained with an inverted microscope (Olympus IX70), using a TILL monochromator as light source. Pictures were taken with an attached cooled CCD camera (Orca II-ER, Hamamatsu).
The following antibodies were used: anti-vinculin monoclonal antibody (Chemicon, ref. MAB3574) and anti-Profilin polyclonal and monoclonal antibodies (Cell Signaling, ref. 3237 and Synaptic system, ref. 308 011). Secondary antibodies, Oregon green Phalloidin and Phalloidin-Alexa Fluor® 594 were purchased from Invitrogen. Image J analysis software (National Institutes of Health) was used to quantify spreading and focal adhesion.
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10

Quantifying Adherent Cell Stiffness via MTC

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To quantify stiffness of the living adherent cell, we used MTC as described previously50 (link),51 (link). In brief, a functionalized ferrimagnetic microbead bound to the cytoskeleton (CSK) through cell surface integrin receptors was magnetized horizontally and then twisted in a vertically aligned homogenous magnetic field that varied sinusoidally in time. The sinusoidal twisting field causes both a rotation and a pivoting displacement of the bead. As the bead moves, the cell develops internal stresses that in turn resist bead motions. Lateral bead displacements in response to the resulting oscillatory torque were detected via a CCD camera (Orca II-ER, Hamamatsu, Japan) attached to an inverted optical microscope (Leica Microsystems, Bannockburn, IL), and with an accuracy of 5 nm using an intensity-weighted center-of-mass algorithm. We defined the ratio of specific applied torque to lateral bead displacements as the complex elastic modulus of the cell, g*(f) = g’ (f) + i g” (f), where g’ is the storage modulus (cell stiffness), g” is the loss modulus (cell friction), and i2=−1. Cell stiffness and friction are expressed in units of Pascal per nm (Pa/nm). Statistic analyses were performed using unpaired two-tailed Student's t tests. To satisfy the normal distribution assumptions, cell stiffness data were square root transformed and confirmed by the Jarque-Bera test.
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