Since its invention 29 years ago, two‐photon laser‐scanning microscopy has evolved from a promising imaging technique, to an established widely available imaging modality used throughout the biomedical research community. The establishment of two‐photon microscopy as the preferred method for imaging fluorescently labelled cells and structures in living animals can be attributed to the biophysical mechanism by which the generation of fluorescence is accomplished. The use of powerful lasers capable of delivering infrared light pulses within femtosecond intervals, facilitates the nonlinear excitation of fluorescent molecules only at the focal plane and determines by objective lens position. This offers numerous benefits for studies of biological samples at high spatial and temporal resolutions with limited photo‐damage and superior tissue penetration. Indeed, these attributes have established two‐photon microscopy as the ideal method for live‐animal imaging in several areas of biology and have led to a whole new field of study dedicated to imaging biological phenomena in intact tissues and living organisms. However, despite its appealing features, two‐photon intravital microscopy is inherently limited by tissue motion from heartbeat, respiratory cycles, peristalsis, muscle/vascular tone and physiological functions that change tissue geometry. Because these movements impede temporal and spatial resolution, they must be properly addressed to harness the full potential of two‐photon intravital microscopy and enable accurate data analysis and interpretation. In addition, the sources and features of these motion artefacts are varied, sometimes unpredictable and unique to specific organs and multiple complex strategies have previously been devised to address them. This review will discuss these motion artefacts requirement and technical solutions for their correction and after intravital two‐photon microscopy.

中文翻译：

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小动物的多光子活体显微镜：运动伪影挑战和技术解决方案

自 29 年前发明以来，双光子激光扫描显微镜已经从一种很有前途的成像技术发展成为一种在整个生物医学研究界广泛使用的成熟成像方式。双光子显微镜作为活体动物荧光标记细胞和结构成像的首选方法的建立可归因于完成荧光产生的生物物理机制。使用能够在飞秒间隔内提供红外光脉冲的强大激光器，促进了荧光分子仅在焦平面上的非线性激发，并由物镜位置决定。这为在高空间和时间分辨率下研究生物样品提供了许多好处，同时具有有限的光损伤和卓越的组织穿透性。事实上，这些特性已经确立了双光子显微镜作为多个生物学领域活体动物成像的理想方法，并导致了一个全新的研究领域，致力于对完整组织和活生物体中的生物现象进行成像。然而，尽管双光子活体显微镜具有吸引人的特点，但本质上受到来自心跳、呼吸周期、蠕动、肌肉/血管张力和改变组织几何形状的生理功能的组织运动的限制。由于这些运动阻碍了时间和空间分辨率，因此必须正确解决它们以利用双光子活体显微镜的全部潜力并实现准确的数据分析和解释。此外，这些运动伪影的来源和特征各不相同，有时对于特定器官是不可预测的和独特的，并且先前已经设计了多种复杂的策略来解决这些问题。本综述将讨论这些运动伪影的要求和校正技术解决方案，以及活体双光子显微镜检查后的技术解决方案。