Cells were plated into eight-well μ-slide chambers (Ibidi) and imaged on a ZEISS Axioscope/Yokagawa X1 spinning-disk confocal microscope with a 20× 0.8 NA air objective at 37°C and 10% CO2 for most experiments. For experiments reported in Figs. S3 G, S2 C, and 1 G , cells were imaged on an Ultraview spinning disk system (PerkinElmer). Acquisition was performed on a Ti inverted microscope stand (Nikon) with Perfect Focus, fitted with a Plan Apochromatic 20×/0.75 dry objective (Nikon). Images were recorded on an ImagEM EMCCD camera cooled to −69°C (Hamamatsu Photonics). Cells were maintained at 37°C and 5% CO2 on a Chamlide stage top incubator (Live Cell Instrument) for the duration of the experiment. Videos were cropped and adjusted for brightness and contrast using ImageJ and Photoshop. Cytoskeleton drugs were added at least 1 h before imaging. For confinement slide imaging, cells were plated in a two-well glass-bottom μ-slide (Ibidi), arrested in S phase for 24 h, treated with drug for 2 h, and confined for 1 h before imaging.
Imagem emccd camera
Manufactured by Hamamatsu Photonics
Sourced in Japan, Germany
The ImagEM EMCCD camera is a high-sensitivity imaging device manufactured by Hamamatsu Photonics. It utilizes electron-multiplying charge-coupled device (EMCCD) technology to capture low-light images and videos with high signal-to-noise ratios. The camera is designed to provide efficient detection and quantification of faint optical signals.
Automatically generated - may contain errors
Lab products found in correlation
Dmi600b microscope, by Leica (3 mentions)
Axio observer z1 inverted microscope, by Zeiss (2 mentions)
Definite focus, by Zeiss (2 mentions)
Plan apo objective na 0.8, by Hamamatsu Photonics (2 mentions)
Cellsens software, by Olympus (2 mentions)
Lv200 bioluminescence microscope, by Hamamatsu Photonics (2 mentions)
Uplansapo, by Hamamatsu Photonics (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Dmi 4000, by Hamamatsu Photonics (2 mentions)
Ultra 888 emccd, by Oxford Instruments (2 mentions)
Scientifica slice scope, by Cairn Research (2 mentions)
Na 1.45 tirf objective, by Hamamatsu Photonics (2 mentions)
Ti e stand, by Yokogawa (2 mentions)
Csu x1 spinning disk head, by Yokogawa (2 mentions)
μ slide eight well chambers, by Ibidi (1 mentions)
Ultraview spinning disk system, by PerkinElmer (1 mentions)
Perfect focus, by Nikon (1 mentions)
Attofluor chamber, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Metamorph 7, by Molecular Devices (1 mentions)
38 protocols using imagem emccd camera
1
Spinning-Disk Confocal Microscopy for Live-Cell Imaging
2
Measuring Cellular Traction Forces on mPADs
3
Yeast Cell Adhesion and Live Imaging
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Yeast cells were grown to logarithmic phase on SC-Trp medium at 25°C. They were adhered to ConA coated coverslips. Cells were incubated for 10 min at room temperature on the ConA coated coverslip and then washed with SC-Trp medium. Cells were imaged on the coverslip in 40 µl of SC-Trp medium. All samples were imaged at room temperature using an Olympus IX81 wide-field epifluorescence microscope equipped with a 100×/1.45 objective. For single channel live cell imaging 488 nm laser light and images were acquired with 80–100 ms exposure time. Emission light was filtered using the GFP-3035C-OMF single-band filter set (Semrock, Rochester, NY). Fluorescence was detected using the Hamamatsu ImagEM EMCCD camera. For two color live cell imaging the samples were excited simultaneously with 488 nm and 561 nm laser light for 250 ms exposure. Excitation light was reflected with a OBS-U-M2TIR 488/561 (Semrock, Rochester, NY) dichroic mirror. Emission light was split and filtered with the DUAL-view (Optical Insights, LLC, Tucson, AZ) beam splitter. The beam splitter created two separated images, one for each channel, on the Hamamatsu ImagEM EMCCD camera sensor. Photobleaching was performed using a custom built setup with a 488 nm laser, focused on a ∼0.5 µm spot.
The wide-field epifluorescence microscope setup was controlled by Metamorph 7.5 (Molecular Devices, Sunnyvale, CA).
The wide-field epifluorescence microscope setup was controlled by Metamorph 7.5 (Molecular Devices, Sunnyvale, CA).
4
Live-cell imaging of nuclear factors
5
Live-cell imaging of nuclear factors
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Samples were imaged on a Zeiss AxioObserver Z1 inverted microscope using a Zeiss 20x plan apo objective (NA 0.8) using the appropriate filter sets and a Hamamatsu ImagEM EMCCD camera. Cells were maintained in a 37 degree incubation chamber at 5% CO2. Cells were imaged every 15 minutes. Focus was maintained using a combination of Zeiss Definite Focus and, using a custom script in MicroManager 2.0 beta (Edelstein et al., 2014 ), software autofocus adjustments every hour to compensate for slight movement of the membrane. For maximum accuracy, cells in this time-lapse were tracked manually in Fiji (Schindelin et al., 2012 (link)) (Figure 2B , n = 40; Figure 5B , FOXB2 transduced, n = 40; Figure 5B FOXB2 nontransduced controls, n = 30; Figure 5B CFP transduced, n = 32; Figure 5B CFP nontransduced controls, n = 44), and the tracks were analyzed with a custom python script that performed illumination profile correction. All mitotic events were captured because we were imaging nuclear transcription factors. Occasionally, a cell track could not be resolved confidently from the beginning to the end of the time-lapse, and any such tracks were truncated to cover only the high-confidence portion of the track.
6
Measuring BRET in HeLa Cells
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HeLa cells were transfected with Nluc/target fusion constructs as described previously (see above). Cells suspended at a density of 2 × 105 cells per ml in DMEM (Gibco)+10% FBS (Hyclone) were plated into 35 mm tissue culture treated imaging dishes (ibidi) at 2 ml per dish and incubated 18–24 h at 37 °C/5% CO2. Media was removed from imaging dishes via gentle aspiration and replaced with 2 ml warm Opti-MEM without phenol red (Gibco) in the presence or absence of tracer +/− competing cold compound. Tracers and cold compound pairs used include 1 μM SAHA-NCT +/− 10 μM SAHA, 3 μM IBET-NCT +/− 10 μM IBET and 3 μM BIBF-1120-NCT +/− 10 μM Nilotinib. Cells were equilibrated for 2 h at 37 °C+5% CO2 before addition of NanoBRET NanoGlo Substrate. Images were captured on an Olympus LV200 microscope equipped with an environmental stage and a Hamamatsu ImagEM EMCCD camera. All images were acquired using a × 100/1.4 UPLanSApo objective and Olympus cellSens software. To image BRET events, images of donor and acceptor emission were acquired sequentially using a 460/80 bandpass filter and a 590 nm long-pass filter, respectively.
7
Spinning Disc Confocal Microscopy Imaging
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Images were acquired on a DMI600B microscope (Leica) with an ImagEM EM-CCD Camera (Hamamatsu Photonics, Hamamatsu, Japan) in a spinning disc confocal setup (Yokogawa). Imaging was done using Micro-Manager 1.4 Software (http://www.micro-manager.org ). Assembling of frames into a single image was performed in ImageJ.
8
Live Cell Imaging of Artificial LDs
9
Immunohistochemical analysis of 5mC and 5hmC
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Growing oocytes collected at postnatal day 7 were fixed in 3.7% paraformaldehyde in PBS for 20 min, washed with PBS containing 0.1% BSA, permeabilized with 0.5% Triton X-100 for 15 min. The cells were denatured with 4 N HCl for 10 min, neutralized with 100 mM Tris-HCl (pH 8.5) for 20 min, and then incubated with 1/500 anti-5mC (Eurogentec) and 1/500 anti-5hmC (Active Motif) primary antibodies for 1 h at room temperature. After washing with in PBS with BSA, the cells were incubated with 1/250 fluorescein isothiocyanate-conjugated anti-mouse IgG (Jackson Immuno-Research) and 1/250 rhodamine-conjugated anti-rabbit IgG (Jackson Immuno-Research) for 1 h. The oocytes were then mounted on a glass slide in VECTASHEILD medium with DAPI (Vector Laboratory) and observed under a CSU-10 confocal laser scanning microscope (Yokogawa) with an ImagEM EM-CCD camera (Hamamatsu).
10
Imaging Immobilized UQ-Body and Antigen
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To image the immobilized UQ-body on the beads, 10 μL of Anti-Flag M2 affinity gel and 100 ng of double ATTO520-labeled UQ-body were incubated for 30 min at room temperature on a rotating wheel. After washing with PBS to remove unbound UQ-body, 0–1 μg of BGP-C7 was added. The sample was subsequently placed on a 35-mm glass bottom dish, and imaged using a fluorescence microscope IX71 (Olympus, Tokyo, Japan) equipped with 470 ± 20 nm excitation and 520 ± 20 nm emission filters. The images were obtained using the HCImage system equipped with an ImagEM EM-CCD camera (Hamamatsu Photonics, Japan).
To image the antigen on the beads, streptavidin agarose beads (10 μL) and 1 μg of biotinylated BGP-C11 peptide were incubated for 30 min at 25°C on a rotating wheel. After washing by centrifugation (1,000 × g, 1 min, 4°C) to remove unbound peptide, 100 ng of double ATTO520 labeled UQ-body was added, followed by washing with PBST. After the addition of 250 μL PBST, the sample was placed on a 3.5-cm dish, and imaged. Controls without biotinylated BGP-C11 peptide or UQ-body were employed.
To image the antigen on the beads, streptavidin agarose beads (10 μL) and 1 μg of biotinylated BGP-C11 peptide were incubated for 30 min at 25°C on a rotating wheel. After washing by centrifugation (1,000 × g, 1 min, 4°C) to remove unbound peptide, 100 ng of double ATTO520 labeled UQ-body was added, followed by washing with PBST. After the addition of 250 μL PBST, the sample was placed on a 3.5-cm dish, and imaged. Controls without biotinylated BGP-C11 peptide or UQ-body were employed.
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