Long-term live imaging, cell identification and cell tracking in regenerating crustacean legs

Area of Science:

• Developmental Biology
• Regenerative Medicine
• Imaging Science

Background:

• High-resolution live imaging of complex biological processes like regeneration is challenging.
• Existing methods struggle with specimen mobility, long durations, and light-induced photodamage.
• Previous work focused on partial imaging of crustacean leg regeneration in *Parhyale hawaiensis*.

Purpose of the Study:

• To develop and present a comprehensive method for live imaging of entire crustacean leg regeneration.
• To achieve cellular resolution over extended periods (up to 10 days) with minimal photodamage.
• To enable detailed cell lineage tracing of regenerated tissues.

Main Methods:

• Optimized mounting and long-term live imaging of regenerating crustacean legs.
• In situ staining of regenerated tissues post-imaging to identify cell fates.
• Computer-assisted cell tracking to reconstruct cell lineages and progenitor relationships.

Main Results:

• A method was established for capturing the complete leg regeneration process at cellular resolution.
• The technique successfully minimizes light exposure while maximizing imaging and tracking efficiency.
• The approach is compatible with cell-type-specific markers for comprehensive lineage analysis.

Conclusions:

• This method overcomes key challenges in live imaging of long-term regeneration.
• It provides a powerful tool for describing cell lineages during limb regeneration.
• Potential applications include detailed mapping of cellular contributions to tissue regrowth.