Practical intravital two-photon microscopy for immunological research: faster, brighter, deeper

Area of Science:

• Immunology
• Cell Biology
• Microscopy

Background:

• Video-rate two-photon microscopy has transformed cell biology by enabling high-speed, deep-tissue imaging of intact samples.
• In immunology, this technique is crucial for understanding immune cell dynamics within secondary lymphoid organs, which dictates immune response effectiveness.

Purpose of the Study:

• To discuss practical aspects of establishing an intravital two-photon microscopy facility for immunology research.
• To highlight how advancements in lasers, optics, and photochemistry enable sophisticated applications.
• To cover new fluorescent dyes, reporter mice, and microsurgical techniques for enhanced imaging.

Main Methods:

• Establishing a basic intravital two-photon microscopy facility.
• Utilizing advances in ultrafast lasers, non-linear optics, and localized photochemistry.
• Employing next-generation fluorescent dyes and reporter mice.
• Applying microsurgical principles for sample preparation.

Main Results:

• Two-photon microscopy is becoming more accessible due to commercial availability and turnkey solutions.
• Advanced applications like photoactivation, photobleaching, spectral fingerprinting, and single-cell tracking are supported by technological progress.
• New reagents and surgical techniques enhance the interrogation of biological processes.

Conclusions:

• Intravital two-photon microscopy is a vital and increasingly accessible tool for immunological research.
• Technological advancements are expanding the capabilities of two-photon microscopy for studying complex biological systems.
• The integration of advanced instrumentation, reagents, and techniques facilitates deeper insights into immune cell behavior and function.