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

Hao Sha, Yu Wu, Yongbing Zhang (), Ran Liu, Xiaochen Feng, Haoyang Li, Zhong Wang, Xiufeng Zhang and Shangguo Hou ()
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Hao Sha: Harbin Institute of Technology (Shenzhen)
Yu Wu: Shenzhen Bay Laboratory
Yongbing Zhang: Harbin Institute of Technology (Shenzhen)
Ran Liu: Shenzhen Bay Laboratory
Xiaochen Feng: Harbin Institute of Technology (Shenzhen)
Haoyang Li: Shenzhen Bay Laboratory
Zhong Wang: Shenzhen Bay Laboratory
Xiufeng Zhang: Shenzhen Bay Laboratory
Shangguo Hou: Shenzhen Bay Laboratory

Nature Communications, 2025, vol. 16, issue 1, 1-11

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-63787-3

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