Intravital imaging of the mouse thymus using 2-photon Microscopy

Area of Science:

• Immunology
• Cell Biology
• Microscopy

Background:

• Two-photon microscopy (TPM) enables deep tissue imaging, crucial for observing cellular behavior in live animals.
• Regulatory T cells (Tregs), particularly thymus-derived naturally-occurring Tregs (nTregs), play vital roles, but their in vivo interactions remain poorly understood.
• Existing imaging techniques are often laborious and technically demanding, limiting the study of cellular dynamics within organs.

Purpose of the Study:

• To describe a novel surgical procedure and stereotactic organ holder for intravital imaging of the thymus.
• To enable the observation of Foxp3+ regulatory T cell movements and interactions within a transplanted thymus.
• To facilitate the study of factors influencing regulatory T cell generation and behavior in vivo.

Main Methods:

• Utilized nude mice, gamma-irradiated and reconstituted with bone marrow from Foxp3-KI(gfp/gfp) mice.
• Transplanted embryonic thymus into the kidney capsule of recipient mice.
• Developed a stereotactic organ holder for stabilizing the kidney-containing thymus for intravital two-photon microscopy under physiological conditions.

Main Results:

• Successfully established a protocol for imaging regulatory T cells within a transplanted thymus in live mice.
• Demonstrated the capability to track Foxp3+ cell dynamics and document their behavior in vivo.
• The developed system allows for the investigation of drug effects on regulatory T cell generation.

Conclusions:

• The described surgical protocol and stereotactic holder provide a powerful new tool for intravital imaging of the thymus.
• This technique overcomes previous limitations in observing cellular interactions within the thymus, particularly for regulatory T cells.
• The method is applicable to studying various aspects of T cell biology and the impact of pharmacological interventions.