Imaging of live and fixed cells was performed using a temperature-controlled spinning disk confocal system from Quorum Technologies, Inc. (Guelph, Canada) on a Nikon Eclipse Ti microscope. Images and movies were processed using Nikon Elements and Photoshop CS (Adobe, San Jose, CA). Further details are in Supplementary Information .
Spinning disk confocal system
Manufactured by Quorum Technologies
Sourced in Canada
The Spinning disk confocal system is a type of microscope that uses a spinning disk with multiple pinholes to rapidly scan a sample and capture high-resolution, real-time images. It provides optical sectioning capability, allowing for the visualization of structures within a sample in a non-invasive manner.
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3 protocols using spinning disk confocal system
1
Spinning Disk Confocal Imaging
2
Spinning Disk Confocal Microscopy Protocol
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Fluorescence images were acquired using spinning disk confocal microscopy. The spinning disk confocal system (Quorum Technologies) used in our laboratory is based on a Zeiss Axiovert 200M microscope (Carl Zeiss) with 63× (numerical aperture [NA] 1.4) or 100× (NA 1.45) oil immersion objective, equipped with diode-pumped solid-state lasers (405, 440, 491, 561, and 655 nm; Spectral Applied Research) and a motorized XY stage (Applied Scientific Instruments). Images were acquired using a back-thinned, electron-multiplied, cooled, charge-coupled device camera (C9100-13 ImagEM; Hamamatsu Photonics) controlled by the Volocity software (PerkinElmer).
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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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