Abstract
Fluorescence spectra offer rich physicochemical insights into molecular environments and interactions. However, imaging the dynamic fluorescence spectrum of rapidly moving biomolecules, along with their positional dynamics, remains a significant challenge. Here, we report a three-dimensional target-locking-based single-molecule fluorescence Spectrum Dynamics Imaging Microscopy (3D-SpecDIM), a method capable of simultaneously capturing both rapid 3D positional dynamics and physicochemical parameter changing dynamics of the biomolecules with enhanced spectral accuracy, high spectral acquisition speed, single-molecule sensitivity, and high 3D spatiotemporal localization precision. As a demonstration, 3D-SpecDIM is applied to real-time spectral imaging of the mitophagy process, highlighting its enhanced ratiometric fluorescence imaging capability. Additionally, 3D-SpecDIM is used for multi-resolution imaging, providing valuable contextual information on the mitophagy process. Furthermore, we demonstrated the quantitative imaging capability of 3D-SpecDIM by imaging the cellular blebbing process. By continuously monitoring the physicochemical parameter dynamics of biomolecular environments through spectral information, coupled with 3D positional dynamics imaging, 3D-SpecDIM offers a versatile platform for concurrently acquiring multiparameter dynamics, providing comprehensive insights unattainable through conventional imaging techniques. This work represents a substantial advancement in single-molecule spectral dynamics imaging techniques.