Single molecule spectrum dynamics imaging with 3D target-locking tracking

  • Nat Commun. 2025 Sep 30;16(1):8686. doi: 10.1038/s41467-025-63787-3.
Hao Sha  #  1  2 ,  Yu Wu  #  2  3 ,  Yongbing Zhang  #  4  5 ,  Ran Liu  2  3 ,  Xiaochen Feng  1 ,  Haoyang Li  2  6 ,  Zhong Wang  2 ,  Xiufeng Zhang  2  6 ,  Shangguo Hou  7
Affiliations
  • 1. School of Computer Science and Technology, Harbin Institute of Technology (Shenzhen), Shenzhen, China.
  • 2. Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, China.
  • 3. School of Life Science and Technology, Harbin Institute of Technology, Harbin, China.
  • 4. School of Computer Science and Technology, Harbin Institute of Technology (Shenzhen), Shenzhen, China. [email protected].
  • 5. Pengcheng Laboratory, Shenzhen, China. [email protected].
  • 6. Center for Ultrafast Science and Technology, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, China.
  • 7. Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, China. [email protected].
  • # Contributed equally.
PMID: 41027930 DOI: 10.1038/s41467-025-63787-3
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.

Products