In Vivo Light-Sheet Imaging of Senescence Reporter Activity in a Transparent Killifish

Area of Science:

• Aging research
• Cellular biology
• Microscopy techniques

Background:

• Cellular senescence accumulates with age, contributing to tissue dysfunction.
• Current methods for senescence assessment require tissue removal, preventing in vivo longitudinal studies.
• A GFP reporter line in transparent killifish (Nothobranchius furzeri) was previously developed.

Purpose of the Study:

• To describe a protocol for in vivo light-sheet microscopy of senescence-associated cell cycle arrest.
• To enable real-time, single-cell resolution monitoring of senescence in a living vertebrate model.
• To facilitate the study of senescence dynamics in short-lived organisms.

Main Methods:

• Utilized a cdkn1a (p21)-driven GFP reporter line in transparent killifish.
• Developed a protocol for anesthetizing and mounting fish for stable light-sheet microscopy.
• Acquired 3D image stacks and processed data to monitor GFP-positive cells in intact, living fish.

Main Results:

• Successfully monitored GFP-positive cells, indicating senescence-associated cell cycle arrest, in intact living killifish.
• Achieved high-resolution volumetric imaging with minimal phototoxicity.
• Demonstrated the feasibility of analyzing senescence dynamics in a short-lived vertebrate model.

Conclusions:

• The described light-sheet microscopy protocol enables intravital imaging of cellular senescence in living killifish.
• This method offers a non-invasive approach for studying senescence dynamics in vivo.
• While currently terminal, the protocol could be adapted for longitudinal studies with ethical approval.