Clatworthy Menna R
Laboratory of Molecular Biology, Department of Medicine, University of Cambridge Research Unit, University of Cambridge, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK,
Methods Mol Biol. 2015;1291:63-75. doi: 10.1007/978-1-4939-2498-1_6.
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.
免疫细胞的主要特征之一是其动态特性。淋巴细胞在血液中于二级淋巴器官和组织之间循环，以最大程度地提高对入侵病原体和组织损伤做出快速且适当免疫反应的可能性。诸如组织学和流式细胞术等传统实验技术极大地增进了我们对免疫细胞的了解，但在过去十年中，双光子显微镜彻底改变了我们探究免疫细胞动态行为的能力，而这一方面对其功能至关重要。双光子显微镜依赖于通过同时应用两个较长波长光的光子来激发荧光团。这使得能够以最小的光损伤进行更大深度的成像。因此，可以对活体组织进行成像，包括淋巴结中的免疫细胞。该技术已被用于探究生发中心反应中发生的事件以及生发中心内细胞之间的相互作用，包括滤泡辅助性T细胞（Tfh）、生发中心B细胞和滤泡树突状细胞（FDC）。在此描述一种方法，通过该方法可以在活体小鼠中对腘窝淋巴结生发中心内Tfh和B细胞之间的相互作用进行成像。
