Serum starved cells were infected with LifeAct-RFP and seeded at a low density on glass bottom plates. Total internal reflection fluorescence (TIRF) microscopy was carried out using a Nikon Eclipse TiE microscope illuminated by an Ar laser (GFP) and a diode laser (RFP). Images were acquired on a Photometrics Evolve EMCCD camera controlled by Nikon NIS-Elements. Actin dynamics were recorded at 30 sec per frame for 20 min. EGF (100 ng/mL) was added at the 5 min time point. To quantitatively measure actin protrusion dynamics, we calculated the expanding regions of actin signal changes relative to the initial area for each time frame. At least 25 – 50 different fields, each containing a minimum of one cell/field were recorded per sample for adequate statistical analysis. The mean areas were measured after converting the images to binary format using ImageJ software.
Evolve emccd camera
Manufactured by Nikon
The Evolve EMCCD camera is a high-performance imaging device designed for scientific and research applications. It utilizes Electron Multiplying Charge-Coupled Device (EMCCD) technology to provide high-sensitivity, low-noise image capture. The Evolve EMCCD camera is capable of capturing images with exceptional signal-to-noise ratio, making it suitable for applications that require the detection of low-light signals.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti e microscope, by Nikon (4 mentions)
Nis element, by Nikon (4 mentions)
Lsm 780, by Zeiss (2 mentions)
Axioobserver with 780 quasar confocal module, by Zeiss (2 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Tyrode s salts, by Merck Group (1 mentions)
Orca fusion digital cmos camera, by Nikon (1 mentions)
Eclipse u2000, by Yokogawa (1 mentions)
9 protocols using evolve emccd camera
1
Quantitative Analysis of Actin Dynamics
2
Confocal Imaging of Biological Samples
3
Rapid Chemotaxis Analysis in Dictyostelium
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HO543, DH1, or LW6 (Gβ null) cells were grown in HL5 medium containing 20 μg/ml G418 and 50 μg/ml hygromycin. Two days before the experiment, 2x105 cells were mixed with an overnight culture of K.a. in 250 μl streptomycin-free HL-5 medium and plated on an SM agar plate. On the day of the experiment, cells were washed off the SM plate with DB buffer, washed once, and resuspended in DB at 2x107 cells/ml. Suitable amount of cells were transferred to LabTek II chambered coverglass (Nalge Nunc) containing DB with 5 μM rapamycin and 0.05% DMSO. For folic acid chemotaxis, Femtotips microcapillary pipettes (Eppendorf) filled with 1 mM folic acid were used. Microscopy for this set of experiments was carried out with a Nikon Eclipse TiE microscope illuminated by an Ar laser (YFP) and a diode laser (RFP). Time-lapse images in bright field, YFP, and RFP channels were acquired by a Photometrics Evolve EMCCD camera controlled by Nikon NIS-Elements. Tracks of cell migration were analyzed in ImageJ to obtain directedness and speed of cells.
4
Rhod-3 Calcium Imaging of Injured Microtissues
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Immediately after injury (day 0) and on day 7 after injury, Rhod-3 Calcium Imaging Kit (Invitrogen) was applied to injured and control tissues according to the manufacturer’s protocol. Briefly, microtissues were incubated with dye solution at RT for 45 min, washed, and incubated another 45 min at RT to allow for ester cleavage. Samples were imaged in Tyrode’s salts with sodium bicarbonate (Sigma). Time-lapse videos of the microtissue’s spontaneous contractions were acquired at either 30 or 15 frames per second on a Nikon Eclipse Ti with a ×4 objective with an Evolve EMCCD camera in a humidified chamber at 37°C and 5% CO2. Regions of interest (ROIs) for the center region (299 μm × 199 μm) and edge region (100 μm × 100 μm) of microtissues selected and the change in intensity over time were measured. A two-frame moving average was taken to reduce noise. The amplitude of the calcium waveform was then calculated. As Rhod-3 is not a ratiometric dye, we normalized to the average amplitude for the center ROI of the control for each imaging session for better comparison between experimental replicates.
5
Fluorescence Microscopy Techniques for Live-Cell Imaging
6
Fluorescence Microscopy Techniques for Live-Cell Imaging
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Growth-phase cells were placed in an eight-well cover slip chamber and allowed to adhere for 10-15 min. Then the media was replaced with 450 μl DB. In order to add 5μM rapamycin in most experiments, firstly 1 μl stock (10 mM in DMSO) was diluted with 200 μl DB, then 50 μl of the solution was added to the 450 μl in the chamber drop wise. Imaging usually started no later than 30 min after DB replacement. For experiments presented in Fig. 3d , cells were incubated with 40 μM LY294002 or 20 μM PP242 or both for 80–100 min in DB before imaging started. And for experiments presented in Fig. 3e,f , Fig. 4e–g and Supplementary Video 11 , cells were treated with 5 μM LatrunculinA in DB for about 30 min.
Zeiss LSM780 single-point laser-scanning microscope (Zeiss AxioObserver with 780-Quasar confocal module; 34-channel spectral, high-sensitivity gallium-arsenide phosphide detectors, GaAsP), was used for confocal image acquisition.
Total Internal Reflection Fluorescence (TIRF) microscopy was carried out with a Nikon Eclipse TiE microscope illuminated by an Ar laser (YFP) and diode laser (mCherry). Images were acquired by a Photometrics Evolve EMCCD camera controlled by Nikon NIS-Elements.
Phase illumination on a Zeiss Observer.Z1 inverted microscope equipped with a 20×/0.3 air objective or a 40×oil objective was used for phase image acquisition.
Zeiss LSM780 single-point laser-scanning microscope (Zeiss AxioObserver with 780-Quasar confocal module; 34-channel spectral, high-sensitivity gallium-arsenide phosphide detectors, GaAsP), was used for confocal image acquisition.
Total Internal Reflection Fluorescence (TIRF) microscopy was carried out with a Nikon Eclipse TiE microscope illuminated by an Ar laser (YFP) and diode laser (mCherry). Images were acquired by a Photometrics Evolve EMCCD camera controlled by Nikon NIS-Elements.
Phase illumination on a Zeiss Observer.Z1 inverted microscope equipped with a 20×/0.3 air objective or a 40×oil objective was used for phase image acquisition.
7
Microscopy Imaging and Processing Protocol
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Images were captured using either a Zeiss AxioSkop 2 MOT compound microscope with a QImaging Retiga 2000R camera and an RGB pancake (QImaging), using QCapture Pro 6.0 software, or a Nikon A1 confocal with a Photometrics Evolve EM‐CCD‐camera running on Nikon elements software. Adobe Photoshop CS6 was used to process images.
8
Quantitative Analysis of Actin Dynamics
9
Photoconversion of Tubulin and Mitochondria
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Photoconversions of tdMaple3-tubulin and Mito-MoxMaple3 were performed using illumination from a Heliophor 89 North light in the epifluorescence pathway by a 405 nm filter, either locally (for tdMaple3-tubulin) or globally (for Mito-MoxMaple3) through an adjustable pinhole in the field diaphragm position for 10~20 s. Samples were imaged either on a Nikon Eclipse U2000 inverted stand with a Yokogawa CSU10 spinning disk confocal head with a Photometrics Evolve EMCCD camera and a 40 × 1.30 N.A. oil lens, or a Nikon W1 spinning disk confocal microscope (Yokogawa CSU with pinhole size 50 µm) with a Hamamatsu ORCA-Fusion Digital CMOS Camera and a 40 × 1.25 N.A. silicone oil lens, all controlled by Nikon Elements software.
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