Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula

Area of Science:

• Developmental Biology
• Cellular Mechanics
• Microscopy Techniques

Background:

• Embryonic development relies on coordinated cell movements for tissue and organ formation.
• Mechanical interactions are increasingly recognized as key regulators of these cellular movements.
• Existing laser ablation methods, like UV laser ablation, have limitations in resolution and penetration for studying deep embryonic tissues.

Purpose of the Study:

• To introduce a novel method for precise, deep tissue ablation in embryos using two-photon microscopy.
• To enable the physical perturbation of specific cell populations and mechanical constraints during development.
• To facilitate the study of cell behavior and tissue organization following targeted ablations.

Main Methods:

• Utilized a two-photon microscope for laser ablations, offering superior axial resolution and penetration depth.
• Demonstrated the method in transgenic zebrafish expressing green fluorescent protein in the axial mesendoderm.
• Performed ablations to sever the axial mesendoderm while preserving adjacent tissues (ectoderm, yolk cell).

Main Results:

• Successfully ablated deep, significant, and spatially well-defined volumes within the embryo.
• Demonstrated the ability to isolate cell populations or relieve mechanical constraints without damaging surrounding structures.
• Observed cell behavior using live imaging before and after ablation to assess developmental responses.

Conclusions:

• The described two-photon microscopy ablation protocol provides a powerful tool for investigating mechanical roles in morphogenesis.
• This method offers versatility, applicable to various cell types, tissues, and developmental stages.
• The technique allows for precise physical perturbations, advancing the study of developmental mechanics in vivo.