Time-lapse imaging of the organoid was performed on a MetaMorph v7.10 software in VivaView Incubator Fluorescence Microscope (Olympus) with an X-Cite eXacte as illumination source. DIC image as well as fluorescent images (PI and calcein) were acquired through UPLSAPO20x objective (0.75 N.A.) onto Orca R2 cooled CCD camera (Hamamatsu). After 30 mins to one hours of adaption, single plane imaging was performed for 1.5–6 hours at 120–320s intervals just after FlaTox or TNF+CHX treatment. ImageJ v1.4.3.67 software (NIH) was used to measure the Mean Intensity of each organoid in hyperstuck image for PI and calcein Intensity analysis.
Orca r2 cooled ccd camera
Manufactured by Hamamatsu Photonics
Sourced in United States
The ORCA-R2 is a cooled CCD camera designed for low-light imaging applications. It features a 1344 x 1024 pixel CCD sensor with high quantum efficiency and low dark current. The camera can operate at frame rates up to 30 frames per second and is equipped with a Peltier cooling system to reduce thermal noise. The ORCA-R2 provides a digital interface for data transfer and camera control.
Automatically generated - may contain errors
Lab products found in correlation
Lambda dg 4 exciter, by Sutter Instruments (3 mentions)
Igor pro, by Wavemetrics (3 mentions)
Axioskop 2 fs microscope, by Zeiss (2 mentions)
Thermobox chamber, by Tokai Hit (2 mentions)
Dc2100, by Thorlabs (2 mentions)
Ti e inverted microscope, by Nikon (2 mentions)
Vivaview incubator fluorescence microscope, by Olympus (1 mentions)
Bodipy 558 568 c12, by Thermo Fisher Scientific (1 mentions)
Csu x1 spinning disk, by Yokogawa (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Ix70 fluorescence microscope, by Olympus (1 mentions)
Emccd camera, by Oxford Instruments (1 mentions)
Ti microscope, by Nikon (1 mentions)
Ab84422, by Abcam (1 mentions)
Ab51081, by Abcam (1 mentions)
Ix81 widefield microscope, by Hamamatsu Photonics (1 mentions)
Sc 8418, by Santa Cruz Biotechnology (1 mentions)
Np 40, by Merck Group (1 mentions)
Eclipse ti e microscope, by Nikon (1 mentions)
12 protocols using orca r2 cooled ccd camera
1
Time-lapse Imaging of Organoid Response
2
Fluorescent Lipid Droplet Staining
3
Fluorescent Visualization of Actin Cytoskeleton
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Cells were fixed with 4% paraformaldehyde, washed and blocked with PBS containing 20% Block Ace (KAC Co., Tokyo, Japan), 0.3% Triton X-100 and 5% BSA. To detect filamentous actin (F-actin), cells were stained with Alexa Fluor 488-conjugated Phalloidin (Thermo Fisher Scientific) for 2 h at room temperature. Nuclei were stained using Hoechst dye (Thermo Fisher Scientific). After washing with PBS, cells were mounted and visualized using an IX70 fluorescence microscope (Olympus, Tokyo, Japan) equipped with an ORCA-R2 cooled CCD camera (Hamamatsu Photonics, Shizuoka, Japan).
4
Microscopy Techniques for Protein Localization
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Preparing anchor-away strains expressing FRB-GFP–tagged proteins for microscopy was performed as described (Haruki et al., 2008 (link)). Briefly, 1 ml of cells was harvested, fixed in 1 ml −20°C methanol for 6 min, and resuspended in TBS/0.1% Tween with DAPI. Fixed, DAPI-stained cells were spotted onto a 2% agarose pad on a glass slide and topped with a cover slip. Samples were imaged for both GFP and DAPI on a Nikon Ti2 microscope with a 100x objective and an ORCA-R2 cooled CCD camera (Hamamatsu).
Confocal microscopy of Sis1-YFP, Cfi1-mKate, and Hsp104-mKate was performed live by allowing low density cultures grown in SCD at room temperature to settle in 96-well glass bottom plates coated with concanavalin A. For treatments, medium was removed and fresh SCD containing the indicated drug was added to the well. Imaging was performed on a Nikon Ti microscope with a 100 × 1.49 NA objective, a spinning disk confocal setup (Andor Revolution) and an EMCCD camera (Andor).
Confocal microscopy of Sis1-YFP, Cfi1-mKate, and Hsp104-mKate was performed live by allowing low density cultures grown in SCD at room temperature to settle in 96-well glass bottom plates coated with concanavalin A. For treatments, medium was removed and fresh SCD containing the indicated drug was added to the well. Imaging was performed on a Nikon Ti microscope with a 100 × 1.49 NA objective, a spinning disk confocal setup (Andor Revolution) and an EMCCD camera (Andor).
5
Electrophysiology and Calcium Imaging in Islet Cells
6
Electrophysiology and Calcium Imaging in Islet Cells
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The electrophysiological measurements were performed in intact islets essentially using the perforated-patch whole-cell technique in the voltage- or current-clamp modes in δ-cells.
Parallel measurements of [Ca2+]i and membrane potential were performed using an Axioskop 2FS microscope (Zeiss, Oberkochen, Germany) equipped with a 40x/0.8 objective, Lambda DG-4 exciter (Sutter Instruments, USA) and Orca-R2 cooled CCD camera (Hamamatsu, Japan). Images were acquired using an open-source Micromanager software (developed at Ron Vale’s lab, UCSF, San Francisco, USA) and processed using ImageJ. Data analysis was performed in Igor Pro (Wavemetrics).
Parallel measurements of [Ca2+]i and membrane potential were performed using an Axioskop 2FS microscope (Zeiss, Oberkochen, Germany) equipped with a 40x/0.8 objective, Lambda DG-4 exciter (Sutter Instruments, USA) and Orca-R2 cooled CCD camera (Hamamatsu, Japan). Images were acquired using an open-source Micromanager software (developed at Ron Vale’s lab, UCSF, San Francisco, USA) and processed using ImageJ. Data analysis was performed in Igor Pro (Wavemetrics).
7
Multicolor Immunofluorescence Staining Protocol
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Cells were fixed for 5 min in 3.7% paraformaldehyde (PFA) (Sigma-Aldrich) followed by 2 min permeabilization with 0.5% NP-40 (EMD Millipore). The coverslips were incubated with blocking solution (1% bovine serum albumin) for 1 h at RT. Primary antibodies used for immune staining were diluted in blocking solution and applied to the coverslips for 1 h at RT; this was followed by three short rinses in PBS. Primary antibodies were as follows: anti-Matr3C (ab84422; Abcam), anti-Matr3N (ab51081; Abcam), and mouse anti-S6K used to detected Hedls (sc-8418; Santa Cruz) at 1:200; rabbit anti-Rck/p54 (A300-461A; Bethyl Laboratories, Montgomery, TX) and mouse anti-PABP-1 (10E10, sc-32318; Santa Cruz) at 1:200. Next the coverslips were incubated with secondary antibodies, diluted 1:1000 in blocking buffer, and conjugated to Alexa Fluor 405, 488, or 546 (Invitrogen, Carlsbad, CA) for 1 h at RT. The coverslips were next stained with 4′,6-diamidino-2-phenylindole and rinsed with PBS before being mounted onto slides using Mowiol mounting media. Images were acquired at RT using a using an Olympus IX81 wide-field microscope with a 40× air objective lens attached to a Hamamatsu Orca-R2 cooled CCD camera. Pearson’s colocalization coefficients were determined using the Coloc2 plug-in in imageJ.
8
Measuring Redox Homeostasis in 3D Cultures
9
Single-Cell Bacterial Growth Dynamics
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We used Nikon Ti-E inverted microscope equipped with Plan Apo 100× phase contrast objective (NA1.45), ORCA-R2 cooled CCD camera (Hamamatsu Photonics), Thermobox chamber (Tokai hit, TIZHB), and LED excitation light source (Thorlabs, DC2100). The microscope was controlled by Micromanager (Edelstein et al., 2014 (link)). In the exponential phase experiments, we monitored 25-30 microchambers in parallel in one measurement and acquired the phase-contrast, RpoS-mCherry fluorescence, and GFP fluorescence images from each position with a 3-min interval. We repeated the time-lapse measurement for each culture condition three times. In the regrowth experiment from the stationary phases, we monitored 150-250 microchambers in parallel with a 3-min interval and acquired only phase-contrast images.
We analyzed the time-lapse images by ImageJ (Schneider et al., 2012 (link)). We extracted the information of cell size (projected cell area), RpoS-mCherry fluorescence mean intensity, and GFP fluorescence mean intensity of individual cells along with division timings on each cell lineage for the exponential phase experiment. We extracted only division timings on each cellular lineage for the regrowth experiments from the stationary phases and used this information for further analysis.
We analyzed the time-lapse images by ImageJ (Schneider et al., 2012 (link)). We extracted the information of cell size (projected cell area), RpoS-mCherry fluorescence mean intensity, and GFP fluorescence mean intensity of individual cells along with division timings on each cell lineage for the exponential phase experiment. We extracted only division timings on each cellular lineage for the regrowth experiments from the stationary phases and used this information for further analysis.
10
Dual-color Fluorescence Microscopy Imaging
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Fluorescence microscopy was performed using an Olympus IX83 microscope equipped with a ×100/NA 1.40 (Olympus) or a ×100/NA 1.49 (Olympus) objective and Orca-R2 cooled CCD camera (Hamamatsu), using Metamorph software (Universal Imaging). Double-color imaging were performed using an Olympus IX81 microscope equipped with a high-speed filter changer (Lambda 10-3; Sutter Instruments) that can change filter sets within 40 ms. Simultaneous imaging of red and green fluorescence was performed using an Olympus IX83 microscope, described above, and an image splitter (Dual-View; Optical Insights) that divided the red and green components of the images with a 565-nm dichroic mirror and passed the red component through a 630/50 nm filter and the green component through a 530/30 nm filter. These split signals were taken simultaneously with one CCD camera, described above. All cells were imaged during the early- to mid-logarithmic phase. Images for analysis of co-localization of red and green signals were acquired using simultaneous imaging (64.5 nm pixel size), described above.
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