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Updated: Aug 1, 2026
Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
Published on: August 23, 2007
Two-photon microscopy for imaging germinal centers and T follicular helper cells
1Laboratory of Molecular Biology, Department of Medicine, University of Cambridge Research Unit, University of Cambridge, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK, mrc38@cam.ac.uk.
Insights
Two-photon microscopy allows real-time imaging of immune cell dynamics in live mice. This technique visualizes interactions between T follicular helper (Tfh) and B cells within lymph nodes, crucial for immune responses.
Area of Science:
- Immunology
- Microscopy
- Cell Biology
Background:
- Immune cells, particularly lymphocytes, are inherently dynamic, circulating between tissues and lymphoid organs to ensure rapid responses.
- Traditional methods like histology and flow cytometry have limitations in capturing dynamic cellular behaviors.
- Two-photon microscopy offers advanced capabilities for in vivo imaging of immune cell functions.
Purpose of the Study:
- To describe a novel method for imaging immune cell interactions in live mice.
- To visualize the dynamic behavior of T follicular helper (Tfh) and B cells within germinal centers.
- To enhance understanding of cellular communication critical for adaptive immunity.
Main Methods:
- Utilizing two-photon microscopy for in vivo imaging of immune cells in lymph nodes.
- Employing fluorophore excitation with simultaneous two-photon absorption for deep tissue imaging.
- Developing a protocol to observe Tfh and B cell interactions within a germinal center response in a live mouse model.
Main Results:
- Demonstrated the feasibility of imaging dynamic immune cell interactions within lymph nodes.
- Provided insights into the spatiotemporal behavior of Tfh and B cells during germinal center responses.
- Showcased the utility of two-photon microscopy in studying cellular dynamics with minimal photodamage.
Conclusions:
- Two-photon microscopy is a powerful tool for interrogating the dynamic nature of immune cells.
- The described method facilitates the study of critical cell-cell interactions in lymphoid microenvironments.
- This approach advances our understanding of immune cell function and response mechanisms.
Abstract:
One of the principle features of immune cells is their dynamic nature. Lymphocytes circulate in the blood between secondary lymphoid organs and tissues in an effort to maximize the likelihood of a rapid and appropriate immune response to invading pathogens and tissue damage. Conventional experimental techniques such as histology and flow cytometry have greatly increased our understanding of immune cells, but in the last decade, two-photon microscopy has revolutionized our ability to interrogate the dynamic behavior of immune cells, a facet so critical to their function. Two-photon microscopy relies on the excitation of fluorophores by simultaneous application of two photons of longer wavelength light. This allows a greater depth of imaging with minimal photodamage. Thus, living tissues can be imaged, including immune cells in lymph nodes. This technique has been used to interrogate the events occurring in a germinal center response and the interactions between cells in the germinal center, including T follicular helper cells (Tfh), germinal center B cells, and follicular dendritic cells (FDC). Herein, a method is described by which the interactions between Tfh and B cells within a germinal center in a popliteal lymph node can be imaged in a live mouse.
