For radial glial ablation, Tg(sox10:nls-Eos);Tg(gfap:NTR-mCherry) embryos were immersed in 15 mM Metronidazole (MTZ) solution in egg water with 1% DMSO starting at 10 h prior to imaging (at 10 hpf or 30 hpf) (Johnson et al., 2016 (link); Smith et al., 2016 (link)). Fresh MTZ solution with Tricaine was applied when embryos were mounted for imaging (at 20 or 40 hpf). Control embryos were immersed in 1% DMSO in egg water. Single-cell laser ablation was performed using a nitrogen-pulsed MicroPoint laser (Andor) attached to the spinning disk confocal system mentioned above, a coumarin dye (wavelength 435 nm) and a 40x water immersion objective. To ablate individual cells, a ROI was first created inside the cell of interest to ensure precise ablation. MicroPoint laser was then fired within the ROI using a laser power between 20 to 40 depending on the location of the cell to ablate. Successful laser ablation was confirmed by the disappearance of the fluorescence of the cell (Lewis and Kucenas, 2014 (link)). Immediately after the ablation, conditions of cells and tissue around the ablation site were carefully examined. Embryos with nonspecific damage caused by excessive laser power were excluded from experiments and/or quantification.
Micropoint laser
Manufactured by Oxford Instruments
Sourced in United Kingdom
The MicroPoint laser is a compact, high-precision laser system designed for a variety of scientific and industrial applications. It features a stable, continuous-wave laser source with a small beam footprint, making it suitable for precise micromachining and material processing tasks.
Automatically generated - may contain errors
Lab products found in correlation
C9100 13, by Hamamatsu Photonics (1 mentions)
1.4 na oil immersion objective, by Nikon (1 mentions)
Volocity acquisition software, by PerkinElmer (1 mentions)
Ultraview, by PerkinElmer (1 mentions)
Axiozoom microscope, by Zeiss (1 mentions)
Spinning disk confocal system, by Quorum Technologies (1 mentions)
6 protocols using micropoint laser
1
Radial Glial Cell Ablation in Zebrafish
2
Mitochondrial Ca2+ Imaging in Skeletal Muscle
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Muscle fibers were electroporated with a plasmid for expression of Cepia3mt. Seven days after, skeletal muscle fibers were isolated and incubated with the membrane permeant form of IP3 (5 μM, D-2-3-O-isopropylidene-6-O-(2-nitro-4,5-dimethoxy)benzyl-myo-inositol-triphosphate-hexakis(propionoxymethyl) ester; Sichem GmbH) during 45 min at 37°C. Fibers were placed in the microscope under the beam of the UV laser (Micropoint laser, Andor, Belfast, Northern Ireland). After acquiring a 20 s baseline at 10 Hz, photorelease of caged IP3 was performed by applying a 435 nm UV laser at 15 Hz to a mitochondrial local spot (~3–4 μm in diameter). Fluorescence signals were recorded at 10 Hz in a spinning disk microscope (PerkinElmer, Waltham, MA/Zeiss, Oberkochen, Germany). The results were processed with the formula (ΔF/F0)*100. The background signal recorded in cell-free regions was used to correct the fluorescence.
3
Targeted Cell Ablation with Laser
4
Quantifying Myosin Flows during Cellularization
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Sqh-GFP embryos were prepared for live imaging and were imaged using a spinning disk confocal microscope (Ultraview; PerkinElmer) with a 60×/1.4 NA oil-immersion objective (Nikon), a 488-nm laser, and an electron-multiplying charge-coupled device camera (C9100-13; Hamamatsu). The microscope was controlled with Volocity acquisition software (Improvision). Ablation was performed using a Micropoint laser (Andor Technology) tuned to 365 nm. For each ablation, a focused laser beam was targeted to the middle of an edge marked by Sqh-GFP to generate a point incision. Time-lapse movies of a single z-slice focused at the level of the invagination front were acquired immediately before and after ablation to measure the movement of surrounding tissues upon release of tension. As a control, ablation was performed at the apical cortex in embryos at cycle 13 anaphase.
Velocity maps of myosin flow during the flow phase and tissue movement immediately after laser ablations were generated using the MATLAB-based software OpenPIV (Taylor et al., 2010 (link)) with a spacing/overlap of 8×8 pixels and an interrogation window size of 32×32 pixels. For the laser-ablation experiments, average velocity map was generated from 24 ablations in eight embryos at approximately 5 min after the onset of cellularization.
Velocity maps of myosin flow during the flow phase and tissue movement immediately after laser ablations were generated using the MATLAB-based software OpenPIV (Taylor et al., 2010 (link)) with a spacing/overlap of 8×8 pixels and an interrogation window size of 32×32 pixels. For the laser-ablation experiments, average velocity map was generated from 24 ablations in eight embryos at approximately 5 min after the onset of cellularization.
5
Photoconversion and Lineage Tracing of Neural Crest Cells
6
Laser-Mediated Nerve Transection in Zebrafish
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Nerve transections were preformed using a nitrogen‐dye (435 nm) pumped MicroPoint laser (Andor technology) connected to a spinning disk confocal system (Quorum Technologies) controlled by MetaMorph as previously published (Lewis & Kucenas, 2014 (link); Gwendolyn M. Lewis & Kucenas, 2013 (link); Morris et al., 2017 (link); Rosenberg et al., 2012 (link)). Injuries were conducted using either a 40X water (NA = 1.1) or 63X water (NA = 0.8) objective. Ablation power ranged from 40 to 60 depending on the size of the nerve, the mounting of the larvae, the age of the larvae, and the age of the nitrogen‐dye. For all experiments, injuries were induced in 1–3 spinal motor nerves within hemisegments 4–16, creating an approximately 10 μm injury. Nerves with injuries larger than 10 μm or without a full transection were not included in analyses. To transect nerves, an ellipse was virtually drawn around the desired injury site on an image of the nerve in MetaMorph. The laser was pulsed within the designated region of interest (ROI) until the nerve was injured. Injuries were confirmed by presence of axonal debris and lack of return of motor neuron fluorescence in the ROI after 20 s. In vivo imaging of transected nerves was conducted as described above. In fish that were fixed for antibody staining following nerve transection, the first 10 nerves in each fish were injured.
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