BPS2026 - Mapping single-molecule nanoscale dynamics: Simultaneous 3D localization and spectroscopy via target locking

Fluorescence spectra provide valuable physicochemical insights into molecular environments and interactions. However, simultaneously imaging the dynamic fluorescence spectra of rapidly moving biomolecules alongside their positional dynamics remains a substantial challenge. To address this, we present three-dimensional target-locking-based single-molecule fluorescence spectrum dynamics imaging microscopy (3D-SpecDIM), a method designed to concurrently capture rapid 3D positional dynamics and physicochemical parameter changes in biomolecules. This technique achieves enhanced spectral accuracy, high acquisition speed, single-molecule sensitivity, and high 3D spatiotemporal localization precision. As a demonstration, 3D-SpecDIM was applied to real-time spectral imaging of mitophagy, showcasing its improved ratiometric fluorescence imaging capability. Additionally, the method facilitated multi-resolution imaging, offering contextual insights into the mitophagy process. Furthermore, we quantified the cellular blebbing process using 3D-SpecDIM, underscoring its quantitative imaging proficiency. By continuously monitoring physicochemical parameter dynamics through spectral information and integrating 3D positional tracking, 3D-SpecDIM serves as a versatile platform for acquiring multiparameter dynamics, delivering comprehensive insights beyond conventional imaging approaches. This work marks a significant advancement in single-molecule spectral dynamics imaging techniques.