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.
Keywords: biolabeling; organic fluorophores; photoactivatable; photostable; photoswitchable; single-molecule FRET; single-molecule fluorescence microscopy; super-resolution microscopy.
© 2025 The Authors. Co-published by Nanjing University and American Chemical Society.