A pulsed Micropoint N2 laser (Andor) tuned to 365 nm was used to wound the embryonic epidermis. The laser produced 120-μJ pulses with a duration of 2–6 ns. Ten laser pulses were delivered in seven spots along a 14-μm line to generate a wound. Each embryo was wounded only once. Ten laser pulses were delivered at a single spot to release tension at the wound margin, and samples were imaged immediately before and 1.72 s after spot ablation. In sham-irradiated controls, the laser was completely attenuated using a neutral density filter.
Micropoint n2 laser
Manufactured by Oxford Instruments
Sourced in United States
The Micropoint N2 laser is a compact and efficient nitrogen laser designed for various applications. It generates high-energy ultraviolet pulses with a wavelength of 337.1 nm. The laser operates at a repetition rate of up to 20 Hz and produces pulse energies of up to 100 μJ. The Micropoint N2 laser is a versatile tool for a range of scientific and industrial applications that require a reliable and high-performance ultraviolet light source.
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Lab products found in correlation
Revolution xd spinning disk confocal microscope, by Oxford Instruments (2 mentions)
Ixon ultra 897 camera, by Oxford Instruments (2 mentions)
Halocarbon oil 27 700, by Merck Group (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Volocity, by PerkinElmer (1 mentions)
Matlab, by MathWorks (1 mentions)
Ixon ultra 897 emccd camera, by Oxford Instruments (1 mentions)
10 protocols using micropoint n2 laser
1
Laser-Induced Epidermal Wounding in Embryos
2
Live Imaging of Wound Closure
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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.
3
Laser-Based Subcellular Ablations
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Ablations were induced using a pulsed Micropoint N2 laser (Andor) tuned to 365 nm. The laser delivers 120 µJ pulses at durations of 2–6 ns each. For ablation of cell boundaries, 10 consecutive laser pulses were delivered to a single spot along a cell interface. For single-cell wounds, 10 consecutive laser pulses were delivered to each of two spots spaced 2 µm apart on the medial-apical region of the cell of interest. In experiments where local tension was reduced, 10 laser pulses were delivered to a single spot on the medial-apical region of the cell of interest. Cells were re-ablated upon assembly of medial-apical myosin networks. In sham-irradiated controls, cells were targeted with the laser completely attenuated every 60 s to mimic the repeated ablations performed in the corresponding experiments.
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.
5
Laser-Mediated Dissection of Cardioblast-Pericardial Interface
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Laser cuts were created with a pulsed Micropoint N2 laser (Andor) tuned to 365 nm. The laser delivers 120 µJ pulses of 2-6 ns each. For ablation of the supracellular actin cable at the interface between cardioblasts and pericardial cells, 10 consecutive pulses were delivered at discrete points ~ 2 µm apart along two 14 µm lines perpendicular to the cable. For cuts along the leading edge of the epidermis, 10 consecutive pulses were delivered at discrete points ~ 2 µm apart along a 14 µm line at the boundary between the amnioserosa the epidermis. For controls, the laser was fully attenuated using a neutral density filter.
6
Laser-Mediated Dissection of Cardioblast-Pericardial Interface
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Laser cuts were created with a pulsed Micropoint N2 laser (Andor) tuned to 365 nm. The laser delivers 120 µJ pulses of 2-6 ns each. For ablation of the supracellular actin cable at the interface between cardioblasts and pericardial cells, 10 consecutive pulses were delivered at discrete points ~ 2 µm apart along two 14 µm lines perpendicular to the cable. For cuts along the leading edge of the epidermis, 10 consecutive pulses were delivered at discrete points ~ 2 µm apart along a 14 µm line at the boundary between the amnioserosa the epidermis. For controls, the laser was fully attenuated using a neutral density filter.
7
Laser-Induced Wound Imaging in Embryos
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All wounds were generated with a pulsed nitrogen N2 Micropoint laser (Andor Technology Ltd., Concord, MA, USA) tuned to 435 nm and focused on the cortical surface of the embryo. A region of interest was selected in the lateral midsection of the embryo and ablation was controlled by MetaMorph. On average, ablation time was less than 3s, and time-lapse imaging was initiated immediately. Occasionally, a faint grid pattern of fluorescent dots is visible at the center of wounds that arises from damage to the vitelline membrane that covers embryos.
8
Laser-Induced Embryo Wounding
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All wounds were generated with a pulsed nitrogen N2 Micropoint laser (Andor Technology) tuned to 435 nm and focused on the cortical surface of the embryo. A region of interest was selected in the lateral midsection of the embryo, and ablation was controlled by MetaMorph. On average, ablation time was <3 s, and time-lapse imaging was initiated immediately. Occasionally, a faint grid pattern of fluorescent dots is visible at the center of wounds that arises from damage to the vitelline membrane that covers embryos.
9
Laser-Induced Embryo Wounding Protocol
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All wounds were generated with a pulsed nitrogen N2 Micropoint laser (Andor Technology Ltd.) tuned to 435 nm and focused on the cortical surface of the embryo. A region of interest was selected in the lateral midsection of the embryo, and ablation was controlled by Volocity (PerkinElmer) or MetaMorph (Molecular Devices). The mean ablation time was less than 3 s, and time-lapse imaging was initiated immediately. Occasionally, a faint grid pattern of fluorescent dots is visible at the center of wounds that arises from damaged vitelline membrane that covers embryos.
10
Laser Ablation of Mouse Embryo Cells
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