CHO-K1 hIR/GLUT4-myc-GFP cells were grown and treated the same way as for objective-type TIRF imaging with the following modifications. The cells were incubated with the test substances for 10 min, and then GPMV formation was initiated. After the cells were washed three times with GPMV buffer, they were incubated in GPMV-forming buffer for 1 h. All incubation steps were performed in a humidified atmosphere (≥95%) at 37 °C and 5% CO2. Finally, the buffer with formed GPMVs was transferred into empty wells of a 96-well imaging plate. The imaging was performed on the abovementioned Olympus IX-81 inverted microscope equipped with an IX2-DSU confocal unit. A light guide-coupled illumination system (Olympus U-HGLGPS, Tokyo, Japan) with appropriate filters (FITC) was used to image GFP fluorescence. The fluorescence signal was recorded by an Orca EM-CCD camera (Hamamatsu Photonics, Herrsching, Germany).
Orca em ccd camera
Manufactured by Hamamatsu Photonics
Sourced in Germany
The Orca EM-CCD camera is a high-performance, low-noise imaging device designed for scientific applications. It utilizes an electron-multiplying charge-coupled device (EM-CCD) sensor to provide enhanced sensitivity and improved signal-to-noise ratio, making it suitable for capturing low-light images and videos.
Automatically generated - may contain errors
4 protocols using orca em ccd camera
1
Imaging Giant Plasma Membrane Vesicles
2
Live Imaging of Zebrafish Embryos
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For live imaging, embryos were collected for 15 min to 1 hr, depending on the stage to be imaged, dechorionated with 4.2% sodium hypochlorite for 2 minutes, washed with dH2O, mounted as a monolayer of embryos in a drop of halocarbon oil (Series 56) on the outer surface of the Greiner Lumox culture dishes (Sigma-Aldrich Cat# Z376744), and covered with a coverslip before imaging. All movies were captured using a 40x Planapochromat objective (NA 1.5) on a Nikon Eclipse TE2000-E inverted microscope with a Perkin-Elmer spinning disk and a Hamamatsu Orca-EM CCD camera. Movies were processed using the Metamorph software package (Universal Imaging, Downington, Pennsylvania).
3
Live Imaging of Embryos
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For live imaging, embryos were collected for 15 min to 1 hour, depending on the stage to be imaged, dechorionated with 4.2% sodium hypochlorite for 2 min, washed with dH2O, mounted as a monolayer of embryos in a drop of halocarbon oil (series 56) on the outer surface of the Greiner Lumox culture dishes (Sigma-Aldrich, catalog no. Z376744), and covered with a coverslip before imaging. All movies were captured using a 40× Planapochromat objective [numerical aperture (NA) 1.5] on a Nikon Eclipse TE2000-E inverted microscope with a Perkin-Elmer spinning disk and a Hamamatsu Orca-EM CCD camera. Movies were processed using the Metamorph software package (Universal Imaging, Downington, Pennsylvania).
4
Lipid Droplet Dynamics in Adipocytes
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Differentiated 3T3-L1 adipocytes were treated for 1 and 3 days with the indicated substances (BBR: 3.70 µg/mL; herbal extracts: 10 mg/L) or left untreated. Cells were then stained with LD540 (0.5 µg/mL) for 5 min, washed twice with PBS and incubated for 40–60 min in label-free medium. This step ensured that fluorescence reached a steady-state distribution in the cell. Cells were imaged on an Olympus IX-81 inverted microscope (Olympus, Tokyo, Japan) equipped with an IX2-DSU confocal unit. A light guide-coupled illumination system (Olympus U-HGLGPS) with appropriate filters was used to image LD540 fluorescence. The fluorescence signal was recorded by an Orca EM-CCD camera (Hamamatsu Photonics, Herrsching, Germany). For FRAP experiments, single LDs were photobleached with an intense laser pulse (405 nm) applied for 1,000 ms. Recovery images were recorded at the indicated time intervals. FRAP images were initially analyzed using the built-in FRAP module of the Xcellence RT software. Data were normalized with the pre-bleached image and curve fitting was performed using GraphPad Prism software (version 7). The resulting FRAP curves were plotted based on the standard error of the mean (SEM) and fitted using a mono-exponential equation. The kinetic FRAP parameters were directly obtained from curve fitting.
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