Stage 14–15 embryos were dechorionated in 50% bleach for 2 min, aligned with their ventral-lateral side up on an apple juice agar pad, and transferred to a coverslip coated with heptane glue. Embryos were covered with 1:1 halocarbon oil 27:700 (Sigma-Aldrich) and imaged at 25°C using a Revolution XD spinning disk confocal microscope (Andor Technology) with an iXon Ultra 897 camera (Andor Technology), a 60× oil-immersion lens (NA 1.35; Olympus), and Metamorph software (Molecular Devices). 16-bit Z-stacks were acquired at 0.3-µm steps every 15–60 s and projected for wound closure analysis (15 slices/stack). Wounds were created using a pulsed Micropoint N2 laser (Andor Technology) tuned to 365 nm.
Revolution xd spinning disk confocal microscope
Manufactured by Oxford Instruments
The Revolution XD spinning disk confocal microscope is a high-performance imaging system designed for live-cell and biological sample imaging. It combines a spinning disk confocal unit with a high-speed camera and advanced illumination options to provide fast, high-resolution imaging of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Ixon ultra 897 camera, by Oxford Instruments (2 mentions)
Micropoint n2 laser, by Oxford Instruments (2 mentions)
Halocarbon oil 27 700, by Merck Group (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Na1.2w corr uplanapo objective, by Oxford Instruments (1 mentions)
Dragonfly spinning disk confocal microscope, by Oxford Instruments (1 mentions)
Axio vert a1 inverted microscope, by Zeiss (1 mentions)
4 protocols using revolution xd spinning disk confocal microscope
1
Live Imaging of Wound Closure
2
Time-lapse Photo-conversion Analysis
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Photo-conversion was completed manually with Andor Revolution XD spinning Disk confocal microscope as described above. The time-lapse data were analyzed with Fiji. The pixel values (fluorescent intensity, or FI) of 488 nm and 560 nm channels within selected areas were measured at each time point. The background FI was subtracted to obtain the net FI which was plotted over time to generate the photo-conversion plot. The ratio of net FI (560 nm) over net FI (488 nm) was calculated and plotted over time to generate the conversion rate plot (Figure 3—source data 1 ).
3
Super-resolution Confocal Imaging Analysis
4
Laser Ablation and Cytoskeleton Dynamics
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We conducted laser ablation using a pulsed Micropoint N2 laser tuned to 365 nm and images were captured on a Revolution XD spinning-disk confocal microscope (Andor) using a 60x (NA 1.35) oil immersion lens (Olympus) and an iXon Ultra 897 camera (Andor). Stacks were acquired immediately before and after ablation and every 3 s thereafter for 60 s. Images in which only a single cell junction were cut were analyzed using SIESTA v 4.0 (Fernandez-Gonzalez and Zallen, 2011 (link)). We measured recoil velocity (indicative of relative tensile forces) based on the displacement of vertices at the ends of severed junctions in the first frame captured after cutting. Viscosity-elasticity ratios were estimated using a Kelvin-Voigt model to represent junctions (Fernandez-Gonzalez et al., 2009 (link)). According to this model, the viscosity-to-elasticity ratio is given by the relaxation time for the vertex displacements after ablation. The relaxation time (τ) was calculated by fitting junction retraction to equation L(t) = D(1 – et/τ), where L(t) is the distance between vertices at time t after ablation, and D is the asymptotic distance retracted, proportional to the stress-to-elasticity ratio.
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