多光子荧光显微镜 (MPFM) 已经成为光学成像领域的游戏规则改变者，特别是在研究生物体深处的生物组织方面。 MPFM 通过利用非线性激励将荧光发射主要限制在显微镜焦点体积内，克服了异质组织中光的强散射。这使得能够在完整组织深处进行高分辨率成像，并为结构和功能研究开辟了新途径。 MPFM 已得到广泛的应用，并带来了大量的科学发现和对复杂生物过程的见解。如今，MPFM 已成为许多研究社区不可或缺的工具。它的多功能性和有效性使其成为研究人员在其天然环境中研究细胞和亚细胞水平生物现象的首选技术。在这篇综述中，介绍了 MPFM 的原理、实现、功能和局限性。详细回顾了 MPFM 的三个应用领域：神经科学、癌症生物学和免疫学，并作为将 MPFM 应用到生物研究的示例。版权所有 © 2024 Elsevier Inc. 保留所有权利。

Multiphoton fluorescence microscopy (MPFM) has been a game-changer for optical imaging, particularly for studying biological tissues deep within living organisms. MPFM overcomes the strong scattering of light in heterogeneous tissue by utilizing nonlinear excitation that confines fluorescence emission mostly to the microscope focal volume. This enables high-resolution imaging deep within intact tissue and has opened new avenues for structural and functional studies. MPFM has found widespread applications and has led to numerous scientific discoveries and insights into complex biological processes. Today, MPFM is an indispensable tool in many research communities. Its versatility and effectiveness make it a go-to technique for researchers investigating biological phenomena at the cellular and subcellular levels in their native environments. In this Review, the principles, implementations, capabilities, and limitations of MPFM are presented. Three application areas of MPFM, neuroscience, cancer biology, and immunology, are reviewed in detail and serve as examples for applying MPFM to biological research.Copyright © 2024 Elsevier Inc. All rights reserved.