Stimulated Emission Depletion (STED) imaging was performed using a STEDYCON (Abberior Instruments) with excitation lasers at 450 nm, 594 nm, and 640 nm and a STED laser at 775 nm wavelength (all pulsed), and it was controlled by its own software, Smart Control (SN: SY210901). The STEDYCON was mounted at the camera port of a Zeiss Axio Observer Inverted microscope equipped with a ×100/1.40 oil, UPlanSApo objective. The excitation wavelength range was set to 640 nm for Lifeact-Halo. Depletion power was set to achieve a resolution of 40 nm, the pinhole was set at 64 µm, and the t-series was set to 100 frames with 5 s intervals. Data was stored in obf format and exported as tiff files for further analysis. All images were obtained using LAS AF software (Leica).
Stedycon
Manufactured by Zeiss
Sourced in Germany
STEDYCON is a high-precision and stable optical table from Zeiss. It is designed to provide a stable and vibration-free surface for sensitive optical experiments and setups. The table features a rigid, granite-based construction and advanced damping technology to minimize the effects of external vibrations and disturbances.
Automatically generated - may contain errors
Lab products found in correlation
Stedycon, by Abberior (2 mentions)
Las af software, by Leica (1 mentions)
Axio observer inverted microscope, by Zeiss (1 mentions)
Axio imager, by Zeiss (1 mentions)
Mount liquid, by Abberior (1 mentions)
Mouse monoclonal anti vinculin v9131, by Merck Group (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
Axio imager z2 upright microscope, by Zeiss (1 mentions)
4 protocols using stedycon
1
Super-Resolution STED Imaging of Cells
2
Synthesis and Characterization of FITC-Labeled Collagen III
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The FITC-rhCol III was synthesized by mixing of rhCol III and FITC in a saturated sodium bicarbonate solution for 24 h. The solution was dialyzed in the dialysis tubing (3.5 kD) for 7 days and then freeze-dried. The preparation of FITC-rhCol III coatings was the same, but rhCol III was replaced with FITC-rhCol III. The samples were immersed in PBS on the shaker at 37°C and removed after 7, 14 and 28 days to observe the distribution of FITC-rhCol III under a laser-scanning confocal microscope (LSM880 Airyscan with STEDYCON, Carl Zeiss, Germany).
3
Fn Reorganization Imaging in Cells
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To analyze the reorganization of Fn within the films, substrates were built using fluorescently labeled Fn (Fn*) with Alexa Fluor 568. A total of 5,000 HGF/cm2 were seeded on the samples using complete medium and incubated at 37°C and 5% CO2 during 2, 4, and 7 days, respectively, and immunostained as previously described (Section 2.7.5 ). For STED microscopy experiments, mouse monoclonal anti-vinculin V9131 (Sigma-Aldrich) diluted 1:400 in PBT (PBS + 0.1% Tween 20 + 2% BSA) was used to control focal adhesion formation, and the samples were incubated for 45 min at room temperature. Mouse Abberior STAR RED (Abberior, Göttingen, Germany) diluted 1:200 in PBT was added to the samples as secondary antibody and incubated for 1 h at room temperature. Substrates were washed with PBS (3×) for 15 min and mounted on microscope slides using Abberior Mount Liquid. Samples were examined using Abberior STEDYCON attached to a Zeiss Axio Imager.
4
Multicolor STED Imaging of Cellular Structures
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Expanded cells were imaged with a UPlanSApo 100×/1.4 oil immersion objective in an Olympus IX81 microscope (Olympus, Tokyo, Japan). Z-stacks were deconvolved using the DeconvolutionLab plugin. Stimulated Emission Depletion (STED) imaging was performed using a STEDYCON (Abberior Instruments GmbH, Göttingen) with excitation lasers at 488, 561, and 640 nm, and a STED laser at 775 nm (maximum intensity 1.25 W; all lasers are pulsed with 40 MHz repetition rate). The STEDYCON was mounted on the camera port of an AxioImager Z2 upright microscope (Zeiss, Jena, Germany), equipped with a 100x objective (alpha Plan-Apochromat, Oil, DIC, Vis, NA 1.46; Zeiss). The pinhole was set to 1.1 Airy units for 650 nm emission. Fluorescence was detected on avalanche photo diodes, with emission bands between 650-700 nm, 578-627 nm, and 505-545 nm, respectively. Data was stored in .obf format and exported as tif files for further analysis.
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