Zhang Lei, Shen Dongwen, Yang Jiazhen
School of Life Science and Technology, Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing, 210000, China.
Chem Biomed Imaging. 2025 Apr 1;3(9):572-598. doi: 10.1021/cbmi.5c00007. eCollection 2025 Sep 22.
Recent advancements in single-molecule biophysics have been driven by breakthroughs in advanced fluorescence microscopy techniques and the development of next-generation organic fluorophores. These cutting-edge fluorophores, coupled through tailored biolabeling strategies, offer single-molecule brightness, photostability, and phototunability (i.e., photoswitchable, photoactivatable), contributing to enhancing spatial and temporal imaging resolution for studying biomolecular interactions and dynamics at single-event precision. This review examines the progress made over the past decade in the development of next-generation fluorophores, along with their site-specific labeling methods for proteins, nucleic acids, and biomolecular complexes. It also explores their applications in single-molecule fluorescence-based dynamic structural biology and super-resolution microscopy imaging. Furthermore, it examines ongoing efforts to address challenges associated with fluorophore photostability, photobleaching, and the integration of advanced photophysical and photochemical functionalities. The integration of state-of-the-art fluorophores with advanced labeling strategies aim to deliver complementary correlative data, holding promise for revolutionizing single-molecule biophysics by pushing the boundaries of temporal and spatial imaging resolution to unprecedented limits.
单分子生物物理学的最新进展得益于先进荧光显微镜技术的突破以及下一代有机荧光团的开发。这些前沿荧光团通过定制的生物标记策略相结合，具备单分子亮度、光稳定性和光可调性（即可光开关、光激活），有助于提高空间和时间成像分辨率，从而以单事件精度研究生物分子相互作用和动力学。本综述探讨了过去十年中下一代荧光团的开发进展，以及它们对蛋白质、核酸和生物分子复合物的位点特异性标记方法。还探讨了它们在基于单分子荧光的动态结构生物学和超分辨率显微镜成像中的应用。此外，研究了为应对与荧光团光稳定性、光漂白以及先进光物理和光化学功能整合相关的挑战而正在进行的努力。将最先进的荧光团与先进标记策略相结合旨在提供互补的相关数据，有望通过将时间和空间成像分辨率的边界推向前所未有的极限，给单分子生物物理学带来变革。
