Nonlinear microscopy is a technique that utilizes nonlinear interactions between light and matter to image fluorescence and scattering phenomena in biological tissues. Very high peak intensities from focused short pulsed lasers are required for nonlinear excitation due to the extremely low probability of the simultaneous arrival of multiple photons of lower energy to excite fluorophores or interact with selective structures for harmonic generation. Combined with reduced scattering from the utilization of longer wavelengths, the inherent spatial confinement associated with achieving simultaneous arrival of photons within the focal volume enables deep imaging with low out-of-focus background for nonlinear imaging. This review provides an introduction to the different contrast mechanisms available with nonlinear imaging and instrumentation commonly used in nonlinear microscopy. Furthermore, we discuss some recent advances in nonlinear microscopy to extend the imaging penetration depth, conduct histopathological investigations on fresh tissues and examine the molecular order and orientation of molecules using polarization nonlinear microscopy.

非线性显微镜是一种利用光与物质之间的非线性相互作用对生物组织中的荧光和散射现象进行成像的技术。非线性激发需要来自聚焦短脉冲激光器的非常高的峰值强度，因为多个低能量光子同时到达以激发荧光团或与选择性结构相互作用以产生谐波的概率极低。结合因使用更长波长而减少的散射，与实现光子在焦体积内同时到达相关的固有空间限制使深度成像能够实现非线性成像的低离焦背景。本综述介绍了非线性显微术中常用的非线性成像和仪器的不同对比度机制。此外，我们讨论了非线性显微镜的一些最新进展，以扩展成像穿透深度，对新鲜组织进行组织病理学研究，并使用偏振非线性显微镜检查分子的分子顺序和方向。