For fluorescence staining of SFB, purified mSFB from monocolonized mice were stained for 5 min with DAPI (0.2 mg/ml) and the membrane dye FM4-64FX (5 mg/ml) (Thermo Fisher F34653). Samples were washed twice in PBS and fixed in 4% PFA for 15 min at RT. Samples were again washed twice with PBS, spotted onto slides, mixed with Floromount-G mounting medium (Abcam ab104135), covered with a No. 1.5H cover slip and sealed with nail polish. For structured illumination microscopy acquisition, images (13 images/plane/channel) were taken on a Zeiss LSM780 Elyra PS1 microscope (Carl Zeiss, Germany) using a 63x/1.4 oil Plan Apo objective and an EMCCD Andor Ixon 887 1 K camera. Images were analyzed with ZEN and ImageJ software while the SIMcheck plugin in ImageJ was used to evaluate the acquisition and processing parameters. Brightness/contrast was applied to reduce the background signal.
Lsm780 elyra ps1 microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss LSM780 Elyra PS1 microscope is a high-performance confocal laser scanning microscope designed for advanced imaging applications. It features a modular design, allowing for customization to meet specific research needs. The microscope's core function is to provide high-resolution, three-dimensional imaging capabilities for a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Tetraspeck microsphere, by Thermo Fisher Scientific (4 mentions)
Fm4 64fx, by Thermo Fisher Scientific (2 mentions)
Floromount g mounting medium, by Abcam (2 mentions)
1.518 refractive index oil, by Zeiss (2 mentions)
1.518 refractive index oil, by GE Healthcare (2 mentions)
Ixon 887 1 k camera, by Zeiss (2 mentions)
Plan apo objective, by Zeiss (1 mentions)
Fluoromont g, by Thermo Fisher Scientific (1 mentions)
Glass coverslip, by Marienfeld (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Goat anti rat alexa 555, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by AppliChem (1 mentions)
Gelatin coated 8 well μ slides, by Ibidi (1 mentions)
Mab21651, by R&D Systems (1 mentions)
Cystamine, by Merck Group (1 mentions)
9 protocols using lsm780 elyra ps1 microscope
1
Fluorescence Imaging of Segmented Filamentous Bacteria
2
Structured Illumination Microscopy Protocol
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SIM was performed on a Zeiss LSM 780 Elyra PS1 microscope (Carl Zeiss, Germany) using C Plan-Apochromat 63×/1.4 oil objective with a 1.518 refractive index oil (Carl Zeiss). The fluorescence signal is detected on an EMCCD Andor Ixon 887 1 K. Raw images are composed of fifteen images per plane per channel (five phases, three angles), and acquired with a Z-distance of 0.11 μm. Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio. SIM images were processed separately for each channel with ZEN software and then corrected for chromatic aberration using 100-nm TetraSpeck microspheres (ThermoFisher Scientific) embedded in the same mounting media as the sample. The SIMcheck plugin in imageJ was used to analyze the quality of the acquisition and the processing in order to optimize parameters for resolution, signal-to-noise ratio, and reconstruction pattern63 (link).
3
Super-Resolution Imaging of Samples
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SIM was performed on a Zeiss LSM780 Elyra PS1 microscope (Zeiss) using 100X/1.4 oil Plan Apo objective with a 1.518 refractive index oil (GE Healthcare Life Sciences, USA) and an EMCCD Andor Ixon 887 1 K camera for the detection. SIM images were processed with ZEN software and then aligned with ZEN using 100-nm TetraSpeck microspheres (Thermo Fisher Scientific) embedded in the same conditions as the sample.
4
Super-Resolution Imaging of Fibroblasts
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IMR-90 fibroblasts at PN16 and PN35 were cultured on precision glass coverslips thickness No. 1.5 H (Marienfeld Superior). Immunofluorescence was performed as indicated in the previous section, using Fluoromont-G (Thermo fisher Scientific) as mounting medium. SIM was performed on a Zeiss LSM 780 Elyra PS1 microscope (Carl Zeiss, Germany) using 63×/1.4 oil Plan Apo objective with a 1.518 refractive index oil (Carl Zeiss) and an EMCCD Andor Ixon 887 1 K camera for the detection. Fifteen images per plane per channel (five phases, three angles) were acquired with a Z-distance of 0.09 μm to perform 3D-SIM images. Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio. SIM images were processed with ZEN software and then aligned with ZEN using 100-nm TetraSpeck microspheres (ThermoFisher Scientific) embedded in the same conditions as the sample. The SIMcheck plugin in imageJ was used to analyze the acquisition and the processing in order to optimize for resolution, signal to noise ratio, and reconstruction pattern65 (link).
5
Fluorescence Imaging of Segmented Filamentous Bacteria
6
Super-Resolution Imaging of Astrocyte α-Synuclein
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Astrocytes were cultured on glass coverslips with a thickness of 1.5H (Marienfeld Superior). Immunofluorescence was performed as previously described for confocal microscopy using ProLong Gold as mounting media (ThermoFisher Scientific). SR-SIM was performed on a Zeiss LSM780 Elyra PS1 microscope (Carl Zeiss, Germany) using 63x/1.4 oil Plan Apo objective with a 1.518 refractive index oil (GE healthcare life science) and an EMCCD Andor Ixon 887 1K camera for the detection. 15 images per plane per channel (five phases, three angles) were acquired with a Z-distance of 91 nm to perform SR-SIM images. Acquisition parameters were identical for GFAP, α-syn fibrils and Lamp1 between control and ATTO-550 α-syn fibrils (1µM). SIM images were processed with ZEN software and then aligned with ZEN using 100 nm TetraSpeck microspheres (ThermoFisher Scientific) embedded in the same conditions as the sample. SIMcheck plugin in imageJ [8] (link) was used to analyze the acquisition and the processing in order to optimize between resolution/signal to noise ratio/artifacts. The images of SR-SIM are maximum intensity projections of 17 Z-stacks per condition.
Brightness/Contrast was applied identically between conditions and thresholding was made on the mode value of the histogram of α-syn fibrils.
Brightness/Contrast was applied identically between conditions and thresholding was made on the mode value of the histogram of α-syn fibrils.
7
Super-Resolution Microscopy of Trypanosomes
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Trypanosomes expressing the mNG::IFT81 fusion protein were spread on glass coverslips in medium, and SIM was performed on an LSM780 Elyra PS1 microscope (ZEISS) using 100×/1.46 NA oil Plan Apochromat objective and an electron-multiplying charge-coupled device (EMCCD) Andor IXon 887 1-K camera for the detection at Institut Pasteur. 15 images per plane per channel (five phases; three angles) were acquired to perform the SIM imaging. SIM imaging was processed with ZEN software (ZEISS). The SIMcheck plugin (Ball et al., 2015 (link)) in ImageJ (Schneider et al., 2012 (link)) was used to evaluate the acquisition and the processing parameters.
8
CXCR4 Expression in Stimulated HUVECs
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HUVECs were seeded in gelatin-coated 8 well μ-slides (ibidi GmbH, Martinsried, Germany) at a density of 5×103 cells per well. Cells were serum-starved for 24 hours before stimulation with PZ (3 µg/ml) or SDF-1 (50 ng/ml) in serum- and growth-factor reduced medium (containing 5% FCS). After 8 and 24 hours of stimulation cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 and blocked with 1% bovine serum albumin in PBS for 1 hour. Immunofluorescence staining was performed with primary antibody anti-CXCR4 (1∶50, MAB21651, R&D Systems) over night at 4°C followed by incubation with secondary antibody goat-anti-rat-Alexa555 (1∶400, Life Technologies GmbH, Darmstadt, Germany) for 1 hour at room temperature. Samples without primary antibodies served as negative control. Additionally, nuclei were stained with DAPI (1∶1000; AppliChem, Darmstadt, Germany) for 10 min at room temperature. The fluorescence signals were visualized by using a confocal laser scanning microscope (LSM 780 ELYRA PS.1 microscope, Carl Zeiss Microscopy GmbH, Jena, Germany).
9
Paraformaldehyde Fixed Cell Imaging
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The cells were fixed with 2% paraformaldehyde and labelled as described above. For imaging, the samples were put in freshly prepared Tris buffer (pH 8.0) containing 15 mM cystamine (Sigma-Aldrich) that was complemented with colloidal gold particles (100 nm diameter, British Biocell) as fiducials. The samples were imaged using LSM 780/Elyra PS.1 microscope (Carl Zeiss).
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