Home › Cell Biology › Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation
Cell Biology JoVE (Open Access) Citable · DOI

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

DOI: 10.3791/2093-v
What you'll learn
  • ✓ Perform multicolor confocal time-lapse imaging on transgenic zebrafish larvae in vivo.
  • ✓ Execute targeted neuronal cell ablation to study regeneration responses.
  • ✓ Process and separate spectral channels using ImageJ for visualization analysis.
Protocol

Biopharma Insights In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Difficulty
advanced
Total time
~4–6 hours per imaging session (including larval preparation, live imaging acquisition, and image processing)
Model organism
Danio rerio (zebrafish, transgenic larvae)
Biosafety
BSL-1

Steps

1
Select and prepare embryos and larvae for imaging

Identify appropriate transgenic zebrafish embryos or larvae and prepare them for live confocal microscopy. This includes staging, anesthesia, and mounting on imaging substrates to enable visualization of retinal tissue.

▶ 01:01
2
Acquire multicolor in vivo confocal images

Set up and execute multicolor confocal time-lapse imaging to simultaneously visualize multiple fluorescent transgenic markers in living zebrafish retina. Monitor multiple cell types of interest over time.

▶ 03:02
3
Perform spectral separation image processing with ImageJ

Use ImageJ freeware to unmix and separate overlapping spectral channels from multicolor confocal data. Apply appropriate filtering and processing to isolate individual fluorophore signals.

▶ 05:05
4
Execute targeted neuronal cell ablation and monitor response

Perform targeted ablation of specific neuronal cells within the living zebrafish retina using confocal microscopy. Capture time-lapse sequences to visualize stem cell activation and regeneration responses following cell loss.

▶ 08:09
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