Monitoring Recovery of Laser-Ablated Interneuromast Cells in Zebrafish Using Confocal Microscopy

Abstract

Source: Volpe, B. A., et al. Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells. J. Vis. Exp. (2020)

This video demonstrates monitoring interneuromast cell recovery in zebrafish using confocal microscopy, tracking cell death and repair through time-lapse fluorescence imaging.

Protocol

1. Post-ablation imaging and time-lapse microscopy to study regeneration

1. Click on the dropdown menu for “Channels”. In channels, unclick the “DAPI”(4′,6-diamidino-2-phenylindole) track to inactivate the ablation laser. Click on the dropdown menu for “Acquisition mode”.
2. In acquisition mode, click on “Zoom”.
3. Decrease the zoom to 0.7 either by using the slider or by typing in “0.7.”
   1. To ensure successful cell ablation, increase the gain to 900 and fast scan the field of view.
      NOTE: No GFP (Green fluorescent protein)should be visible within the targeted cell or cells. Using the same or similar settings as those described for pre-ablation imaging, capture and save a post-ablation image (Figure 1C). If the image reveals remnants of fluorescent cells or additional cell bodies that needs to be removed, repeat the laser ablation steps to create a visible gap in the string of interneuromast cells.
   2. Inspect the T-PMT (Transmission Photomultiplier Tube) channel image to confirm cellular damage further. Damaged cells will have a granular appearance, and the nuclei will frequently swell or become irregular in shape (Figure 2).
4. After capturing a pre-ablation image, ablating the cells as necessary, and capturing a post-ablation image for each individual stage position, set up the time-lapse microscopy by activating both stage position and time options for image capture. Set the time parameters to 24 h or another desired endpoint and 15 min intervals. Start experiment to acquire images and save the resulting file when complete (on the following day).
5. If larvae are to be further studied or raised, carefully remove them from the agarose using fine forceps, then transfer them into E3 medium without tricaine for recovery. If they are not to be used further, euthanize according to the approved animal protocol (rapid and sustained immersion in an ice bath is a common method) and dispose of them as required by the institution.

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12-well PTFE Printed Slides	Electron Microscopy Sciences	63425-05
15 mM Tricaine stock solution	Sigma	E10521	15 mM Tricaine in reverse osmosis water
1X E3 + 600 µM Tricaine			Dilute 15 mM Tricaine stock 25X in 1X E3 media
1X E3 media			Dilute 60X E3 media to 1X in reverse osmosis water (16.7 ml/L)
60 X E3 media	All components purchased from Sigma-Aldrich		34.4 g Nacl, 1.52 g Kcl, 5.8 g CaCl2.2H2O, 9.8 g MgSO4.7H20 in 1 liter reverse-osmosis water
Bx60 Compound microscope with mercury arc lamp fluorescence	Olympus
LSM 700 confocal microscope equipped with 405 nm, 488 nm, and 555 or 561 nm lasers	Carl Zeiss Microscopy, LLC		A 5 mW 405 nm laser was used for ablation; Ablations and imaging were performed through a 63x Plan-Apochromat objective with an NA of 1.40
FIJI/ImageJ Image processing software	Multiple contributors		Downloadable at https://fiji.sc/
Dissecting needle modified into hair knife	Fisher Scientific	19010	An eyelash was glued onto the end of a wood-handled dissecting needle
Microsoft Excel software	Microsoft Corp.		Google sheets is a no-cost alternative to Microsoft Excel
Glass bottom 35 mm dishes with no. 1.5 coverslip, 20 mm window	Mattek Corporation	P35G-1.5-20-C	35 mm petri dish, 20 mm glass window
Immersol 518F Immersion Oil	Fisher Scientific	12-624-66A
Low Gelling Agarose	Sigma Life Science	A9414-256
Corning Netwell Insert with 74 um Polyester Mesh, 24 mm Insert	Millipore Sigma	CLS3479-48EA
Rstudio software	Rstudio PBC		Downloadable at https://rstudio.com/
SMX-168-BL Stereo microscope	Motic
Transfer Pipette	Fisher Scientific	13-711-7M
ZEN software	Carl Zeiss Microscopy, LLC