Home › Microscopy & Imaging › Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy
Microscopy & Imaging JoVE (Open Access) Citable · DOI

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

DOI: 10.3791/52654-v
What you'll learn
  • ✓ Set up brightfield stereomicroscopy for live zebrafish larval imaging
  • ✓ Perform tail fin amputation and capture regeneration dynamics in real time
  • ✓ Analyze single-cell resolution tissue repair data from time-lapse sequences
Protocol

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Difficulty
intermediate
Total time
~3–4 hours per larva (including mounting, amputation, and 1–2 hour imaging acquisition)
Model organism
Zebrafish (Danio rerio) larvae
Biosafety
BSL-1

Steps

1
Raise zebrafish larvae to experimental stage

Culture and maintain zebrafish larvae to the appropriate developmental stage for tail fin regeneration studies. Standard husbandry and staging protocols are applied.

▶ 01:34
2
Prepare imaging chamber for microscopy

Assemble and set up the imaging chamber with appropriate mounting medium and environmental controls to maintain larval viability during extended observation.

▶ 02:26
3
Mount and image pre-injured larvae

Position uninjured larvae in the imaging chamber and acquire baseline brightfield stereomicroscopy images to document baseline morphology before amputation.

▶ 03:57
4
Perform precise tail fin amputation assay

Execute controlled amputation of the larval tail fin while monitoring under the microscope to standardize the injury for regeneration analysis.

▶ 05:41
5
Mount larva for continuous time-lapse imaging

Reposition the amputated larva in the imaging chamber with stable mounting to enable uninterrupted long-duration microscopy recording.

▶ 06:00
6
Acquire time-lapse image sequences

Capture brightfield stereomicroscopy image stacks at defined intervals over hours to document the complete regeneration dynamics at single-cell resolution.

▶ 07:28
7
Analyze regeneration data and quantify metrics

Process time-lapse image sequences using image analysis software to extract single-cell resolution measurements and regeneration kinetics.

▶ 09:14
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