SIM imaging was performed with a N-SIM based on an inverted microscope (ECLIPSE Ti-E, NIKON), equipped with an oil immersion TIRF objective lens (SR Apo TIRF 100 × , N.A. 1.49, NIKON), a laser system consisting of 405, 488, 561, and 640 -nm diode lasers (LU-NV, NIKON), and an EMCCD camera (iXon3 DU-897E, Andor Technology). SIM imaging with this system is based on a previous report17 (link). Briefly, excitation lasers were coupled to a multimode optical fiber, collimated, and directed to a fused silica linear transmission-phase grating. A shutter in an intermediate pupil plane discarded all diffraction orders except for 0 and ± 1. The three beams were refocused in the back focal plane of the objective lens. The beams produced as diffraction orders + 1 and −1 were focused near the opposing edges of the back focal plane aperture, and the beam produced as order 0 was focused at its center. Three-dimensional data were acquired with five-pattern phases spaced by 2π/5 and three-pattern orientations spaced 60° apart. The acquired images were computationally reconstructed to obtain a high-resolution image with resolutions of ~115 nm in the x- and y-dimensions and ~270 nm in the z-dimension.
Ixon3 du 897e
Manufactured by Oxford Instruments
Sourced in United Kingdom
The IXon3 DU-897E is a scientific-grade, back-illuminated, electron-multiplying CCD (EMCCD) camera designed for low-light and high-speed applications. It features an electron-multiplying gain stage that can amplify signals before they reach the readout electronics, enabling the detection of single photon events. The camera has a sensor resolution of 1024 x 1024 pixels and a sensor size of 8.2 x 8.2 mm.
Automatically generated - may contain errors
Lab products found in correlation
Nis imaging and image analysis software, by Nikon (3 mentions)
Lu nv, by Nikon (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
Ab64623, by Abcam (1 mentions)
Sc 166081, by Santa Cruz Biotechnology (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
N sim system, by Nikon (1 mentions)
N sim microscope, by Nikon (1 mentions)
N storm 4.0 system, by Nikon (1 mentions)
N sim microscope system, by Nikon (1 mentions)
Incubation chamber stage, by Tokai Hit (1 mentions)
Glass bottom dish, by MatTek (1 mentions)
Uplansapo 60x, by Olympus (1 mentions)
P35g 1.5 14 c, by MatTek (1 mentions)
8 protocols using ixon3 du 897e
1
Super-Resolution SIM Imaging Protocol
2
Comprehensive Cardiomyocyte Characterization
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Dissociated CMs were fixed with 4% PFA and stained with troponin T (cTnT, 1:2000, ab64623, Abcam, Cambridge, USA), MYBPC (1:400, sc-166081, Santa Cruz Biotechnology, TX, USA), connexin 43 (Cx43, 1:1000, C6219, Merk), and MYH6 (1:500, R&D Systems, Minneapolis, USA) primary antibodies, followed by labeling with secondary antibodies: Alexa Fluor 488–conjugated anti-goat, anti-mouse, and anti-rabbit antibodies respectively (1:800, A11055, A21202, A21206, Thermo Fisher Scientific). Images were obtained with Nikon N-SIM (super-resolution system microscope equipped with EM CCD camera iXon3 DU-897E (Andor Technology Ltd, Belfast, UK)). In-house-built selective plane illumination microscopy (SPIM) fluorescent imaging system was used to create the 3D images of the reporter CM cluster (Vuornos et al. 2019 (link)).
Cells at day 15 of CM differentiation were fixed in 4% paraformaldehyde and permeabilized by FACS buffer with 0.1% Triton™ X-100. Troponin T antibody diluted in FACS buffer (1:100) as primary antibody and Alexa Fluor 647–conjugated anti-goat (1:800, Thermo Fisher Scientific) as secondary antibody were used for FACS analysis. The resulting data were analyzed using FlowJo v8.5.2.
Cells at day 15 of CM differentiation were fixed in 4% paraformaldehyde and permeabilized by FACS buffer with 0.1% Triton™ X-100. Troponin T antibody diluted in FACS buffer (1:100) as primary antibody and Alexa Fluor 647–conjugated anti-goat (1:800, Thermo Fisher Scientific) as secondary antibody were used for FACS analysis. The resulting data were analyzed using FlowJo v8.5.2.
3
Super-Resolution Imaging of Spindle Pole Bodies
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For SIM analysis, cells were fixed in the desired cell state for 15 min in 4% paraformaldehyde/2% sucrose in PBS solution and washed extensively with PBS. Cells were placed on a glass-bottomed dish as described in the previous section and were kept for the time of imaging in PBS. The samples were imaged on a Nikon N-SIM system equipped with total internal reflection fluorescence Apochromat 100× 1.49 NA oil immersion objective and a single photon–detection electron-multiplying charge-coupled device camera (iXon3 DU-897E; Andor Technology). 488- and 561-nm laser lines were used for excitation of yeGFP and tdTomato/mCherry, respectively, combined with emission band pass filter 520/45 and 610/60. Images were taken sequentially within a small z stack and in consideration to image SPBs close to the coverslip to minimize spherical aberrations. Subsequently, the reconstruction and channel alignment and FWHM measurements were performed with the NIS imaging and image analysis software (Nikon). For the xyz chromatic shift correction, a reference sample with tetraspeck beads was used. Images always show a single stack of the z slices. For the proximity analysis of NPCs close to the mSPB or satellite/dSPB, plot profiles were generated. Signals within a 200-nm diameter around the yeGFP signal peak were counted as colocalizations.
4
Structured Illumination Microscopy Imaging
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Structured illumination microscopy (SIM) imaging was performed under an N-SIM microscope (Nikon) equipped with a 100× oil immersion objective (Nikon, NA 1.49) and an EMCCD (Andor, iXon3 DU-897E). A z-stack of seven layers with a step size of 0.12 μm was acquired at an exposure time of 20 to 200 ms, an EM gain of 300, and a conversion gain of 2.4× for 640- and 561-nm laser lines. The exposure time and laser intensities were adjusted to achieve a good image quality. SIM images were reconstructed by the three-dimensional (3D) Reconstruct Stack program in the N-SIM module. The reconstructed Z-stack image was projected into a 2D image by maximum intensity projection. The cells were imaged in STORM imaging buffer without β-mercaptoethanol.
5
Single-Molecule Localization Microscopy
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All SMLM imaging was performed using the commercial N-STORM 4.0 system (Nikon) operated in TIRF mode through a 100 × 1.49 NA oil-immersion lens. Cells were imaged using a 647 nm excitation laser (~1.125 kW/cm2) and fluorescence captured on a EMCCD camera iXon3 DU-897E (Andor Technology Ltd.) at 25 °C. A quad bandpass emission filter set (Chroma 89902-ET-405/488/561/647 nm) was used to filter the fluorescence collected from the sample. Images were collected at 50 ms and 20 ms integration time for IRIS and dSTORM imaging respectively, and a total of 100,000 frames were typically recorded for image reconstruction.
6
Super-Resolution Imaging of Tagged Proteins
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For SIM analysis, cells were fixed on a glass coverslip for 30 min with 4% paraformaldehyde and 2% sucrose in PBS buffer. After several wash steps with PBS, the coverslips were mounted on glass slides with Prolong Glass mounting medium. A Nikon N-SIM microscope system (equipped with total internal reflection fluorescence Apochromat 100 × 1.49 NA oil immersion objective and a single photon-detection, electron-multiplying, charge-coupled device camera [iXon3 DU-897E; Andor Technology]) was used to image the samples. tdTomato-tagged proteins were imaged using a 561 nm laser combined with emission bandpass filter 610/60. A single stack of nucleus cross-sections was imaged, and images were reconstructed using NIS imaging and image analysis software (Nikon).
7
Imaging Actin Dynamics in Fertilized Embryos
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For live cell imaging, mRNA was injected to oocytes at the metaphase II stage, followed by ICSI or in vitro fertilization, in order to observe nuclear actin dynamics soon after fertilization. The fertilized embryos injected with mRNAs (10 ng/ml of histone H2B-mCherry and 200 ng/ml of nAC-GFP) were transferred to drops of KSOMaa medium on a glass-bottom dish (MatTek) and placed in an incubation chamber stage (Tokai Hit) at 37 C under 6% CO 2 , 5% O 2 and 89% N 2 on an inverted microscope (IX-71, Olympus), equipped with a Nipkow disk confocal microscope (CSU-W1, Yokogawa Electric), EM-CCD (iXon3-DU897E, Andor Technology), and z motor (Mac5000, Ludl Electronic) (Yamagata and Ueda, 2013) . Images in 51 different focal planes with 2 mm intervals were captured using a silicone oil-immersion objective lens (UPlanSApo 60x, Olympus) every 10 min with 488-nm and 561-nm lasers, which was controlled by the MetaMorph software. Images were analyzed using the MetaMorph, Volocity and ImageJ software.
8
Visualizing Microtubule Organizing Centers in Cells
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Cells were arrested for 2.5 hr with 1.5 µg/ml nocadazole and fixed for 15 min in 4% paraformaldehyde/2% sucrose in phosphate-buffered saline (PBS) solution followed by extensive washing in PBS. The cells were immobilized on a concanavalin A (Sigma-Aldrich, MO, USA)- coated 35 mm glass bottom dish (MatTek, P35G-1.5–14C) and maintained in PBS for the duration of the imaging process in PBS. The samples were imaged in the 2D-SIM mode on a Nikon N-SIM system (Tokyo, Japan) equipped with a TIRF Apochromat 100x/1.49 NA oil immersion objective and a single photon detection EM-CCD camera (Andor iXon3 DU-897E; Belfast, UK). The 488 nm and 561 nm laser lines were used for excitation of yeGFP and tdTomato, respectively, combined with emission band pass filter 520/45 and 610/60. Images were taken sequentially within a small z-stack and in consideration of imaging SPBs close to the coverslip to minimise spherical aberrations. Subsequently the reconstruction and channel alignment was performed using the NIS imaging and image analysis software (Nikon). For the xyz chromatic shift correction we used in a reference sample tetraspeck beads in a reference sample. All images show a single stack of the z-slices.
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