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.
Shangguo Hou (Sun,) studied this question.