In the past two decades, significant advances have been made in single-molecule detection which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics, and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges.

中文翻译：

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单分子追踪及其在生物分子结合检测中的应用

在过去的二十年中，单分子检测取得了重大进展，可以在生理条件下实时直接观察工作中的单个生物分子。特别是，单分子跟踪 (SMT) 显微镜的发展使我们能够监测生命系统中单个生物分子的运动路径，揭示支持生命发展的生物分子的定位动力学和运输方式。除了基于相机的传统跟踪技术的能力之外，近年来开发的最先进的 SMT 显微镜可以在跟踪 3D 空间中的单个分子的同时记录荧光寿命。这种多参数检测能力可以为细胞或组织水平的广泛研究打开大门，包括分子相互作用热点的识别和分子间缔合/解离动力学的表征。在这篇综述中，我们讨论了迄今为止开发的各种 SMT 技术，重点是我们最近开发的下一代 3D 跟踪系统，它不仅实现了超高的时空分辨率，而且提供了适合活体动物成像的足够工作深度。我们还讨论了当前 SMT 技术面临的挑战以及应对这些挑战的潜在策略。重点介绍了我们最近开发的下一代 3D 跟踪系统，该系统不仅实现了超高的时空分辨率，而且还提供了适合活体动物成像的足够工作深度。我们还讨论了当前 SMT 技术面临的挑战以及应对这些挑战的潜在策略。重点介绍了我们最近开发的下一代 3D 跟踪系统，该系统不仅实现了超高的时空分辨率，而且还提供了适合活体动物成像的足够工作深度。我们还讨论了当前 SMT 技术面临的挑战以及应对这些挑战的潜在策略。