Laser & Optoelectronics Progress, Volume. 62, Issue 18, 1817005(2025)

Advances in Three-Dimensional Single-Molecule Tracking Microscopy with Closed-Loop Feedback Control (Invited)

Quan Lu1,3, Yu Wu2,3, Ting Mei2,3, Yue Hu1、**, and Shangguo Hou3、*
Author Affiliations
  • 1School of Electronics and Information Engineering, Harbin Institute of Technology, Harbin 150001, Heilongjiang , China
  • 2School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, Heilongjiang , China
  • 3Systems and Physical Biology Institute, Shenzhen Bay Laboratory, Shenzhen 518132, Guangdong , China
  • show less

    Single-molecule tracking (SMT) imaging technology has become a crucial tool in modern biophysics and cell biology, achieving significant advancements in recent years. By monitoring the dynamic behavior of individual molecules in real time, this technique reveals microscopic processes and transient events that are often obscured by traditional ensemble-averaged methods. This paper provides a comprehensive review of the latest research developments in the field of SMT imaging, with a particular focus on three-dimensional (3D) tracking methods based on closed-loop feedback mechanisms. These methods are primarily categorized into structured signal detection and structured excitation scanning, including techniques such as tetrahedral detection, split detection, tetrahedral excitation, orbital scanning, 3D dynamic photon localization, extremum search localization, minimum cross-entropy localization, and asynchronous readout SPAD arrays. The paper also briefly introduces image acquisition-based closed-loop feedback 3D tracking schemes. We systematically compare and analyze various methods in terms of spatial resolution, temporal resolution, tracking range, system stability, and implementation complexity, aiming to provide guidance for technology selection under different application scenarios. Furthermore, the paper explores integration strategies between 3D tracking and environmental background imaging, as well as multimodal fusion approaches combining optical parameters such as fluorescence lifetime, fluorescence spectrum, and polarization to achieve complementary information for in-depth analysis of complex biological events. Finally, the paper discusses future development directions, including multi-target parallel tracking, deep-tissue imaging, AI-assisted closed-loop control, system miniaturization and integration, and photobleaching suppression. The potential applications of these technologies in cancer biology, neuroscience, virology, and other biomedical fields are also explored. This review aims to provide a systematic reference and theoretical foundation for the continued development and practical deployment of single-molecule tracking technologies.

    Keywords
    Tools

    Get Citation

    Copy Citation Text

    Quan Lu, Yu Wu, Ting Mei, Yue Hu, Shangguo Hou. Advances in Three-Dimensional Single-Molecule Tracking Microscopy with Closed-Loop Feedback Control (Invited)[J]. Laser & Optoelectronics Progress, 2025, 62(18): 1817005

    Download Citation

    EndNote(RIS)BibTexPlain Text
    Save article for my favorites
    Paper Information

    Category: Medical Optics and Biotechnology

    Received: May. 16, 2025

    Accepted: Jun. 13, 2025

    Published Online: Sep. 12, 2025

    The Author Email: Yue Hu (huyue@hit.edu.cn), Shangguo Hou (shangguo.hou@szbl.ac.cn)

    DOI:10.3788/LOP251237

    CSTR:32186.14.LOP251237

    Topics