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Published on: April 9, 2014
Practical intravital two-photon microscopy for immunological research: faster, brighter, deeper
Tri Giang Phan1, Andrew Bullen
1Immunology Programme, Garvan Institute of Medical Research, University of New South Wales, New South Wales, Australia. t.phan@garvan.org.au
Insights
Video-rate two-photon microscopy offers high-speed, minimally invasive imaging for cell biology, especially in immunology. This powerful technique is becoming more accessible for studying immune responses in lymphoid organs.
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.
Abstract:
With its emphasis on minimally invasive high-speed imaging of intact tissues at depth, video-rate two-photon microscopy has revolutionized cell biology. This is particularly true in immunology, where the orchestration of cell migration, cell-cell interactions and intracellular signalling events in multiple distinct anatomical compartments within secondary lymphoid organs is fundamental for achieving an effective immune response. Until recently, access to this powerful tool has been limited to a handful of laboratories with the necessary skills and resources to either custom-build or purchase a commercial two-photon microscope. However, with the entry of more commercial vendors into the market and availability of turnkey solutions, two-photon microscopy is now becoming more accessible. Here, we discuss the practical aspects of establishing a basic intravital two-photon microscopy facility specifically for immunological research and how recent advances in ultrafast lasers, non-linear optics and localized photochemistry can be used to build more sophisticated instruments to support applications such as photoactivation and photobleaching, spectral fingerprinting and automated single-cell tracking. In addition, we discuss the next generation of fluorescent dyes and reporter mice and some of the microsurgical principles required to expose the relevant biology to interrogation by two-photon excitation.
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