Visualizing and tracking cells over time in a living organism has been a much-coveted dream before the invention of intravital microscopy. The opaque nature of tissue was a major hurdle that was remedied by the multiphoton microscopy. With the advancement of optical imaging and fluorescent labeling tools, intravital high resolution imaging has become increasingly accessible over the past few years. Long-term intravital tracking of single cells (LIST) under multiphoton microscopy provides a unique opportunity to gain insight into the longitudinal changes in the morphology, migration, or function of cells or subcellular structures. It is particularly suitable for studying slow-evolving cellular and molecular events during normal development or disease progression, without losing the opportunity of catching fast events such as calcium signals. Here, we review the application of LIST under 2-photon microscopy in various fields of neurobiology and discuss challenges and new directions in labeling and imaging methods for LIST. Overall, this review provides an overview of current applications of LIST in mammals, which is an emerging field that will contribute to a better understanding of essential molecular and cellular events in health and disease.

在活体显微镜发明之前，在生物体内随时间可视化和追踪细胞是梦much以求的梦想。组织的不透明性质是多光子显微镜弥补的主要障碍。随着光学成像和荧光标记工具的发展，在过去几年中，越来越多的人可以进行玻璃体内高分辨率成像。在多光子显微镜下对单个细胞（LIST）进行长期活体内跟踪，提供了一个独特的机会来洞悉细胞或亚细胞结构的形态，迁移或功能的纵向变化。它特别适用于研究正常发育或疾病进展过程中缓慢发展的细胞和分子事件，而又不会丢失捕捉快速事件（如钙信号）的机会。这里，我们回顾了2光子显微镜下LIST在神经生物学各个领域的应用，并讨论了LIST标记和成像方法的挑战和新方向。总体而言，本综述概述了LIST在哺乳动物中的当前应用，这是一个新兴领域，将有助于人们更好地了解健康和疾病中必不可少的分子和细胞事件。