A recent challenge in the field of bioimaging is to image vital, thick, and complex tissues in real time and in non-invasive mode. Among the different tools available for diagnostics, nonlinear optical (NLO) multi-photon microscopy allows label-free non-destructive investigation of physio-pathological processes in live samples at sub-cellular spatial resolution, enabling to study the mechanisms underlying several cellular functions. In this review, we discuss the fundamentals of NLO microscopy and the techniques suitable for biological applications, such as two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG-THG), and coherent Raman scattering (CRS). In addition, we present a few of the most recent examples of NLO imaging employed as a label-free diagnostic instrument to functionally monitor in vitro and in vivo vital biological specimens in their unperturbed state, highlighting the technological advantages of multi-modal, multi-photon NLO microscopy and the outstanding challenges in biomedical engineering applications.

中文翻译：

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非线性光学显微镜：从基础知识到实时生物成像中的应用

生物成像领域最近的一个挑战是以非侵入模式实时对重要的、厚的和复杂的组织进行成像。在可用于诊断的不同工具中，非线性光学（NLO）多光子显微镜可以以亚细胞空间分辨率对活体样本中的生理病理过程进行无标记的无损研究，从而能够研究多种细胞功能的机制。在这篇综述中，我们讨论了 NLO 显微镜的基础知识和适合生物应用的技术，例如双光子激发荧光 (TPEF)、二次和三次谐波发生 (SHG-THG) 以及相干拉曼散射 (CRS)。此外，我们还介绍了一些 NLO 成像的最新示例，用作无标记诊断仪器，可在不受干扰的状态下对体外和体内重要生物样本进行功能监测，突出了多模式、多模式的技术优势。光子非线性光学显微镜和生物医学工程应用中的突出挑战。