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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年9月30日

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  • Hao Sha,
  • Yu Wu,
  • Yongbing Zhang,
  • Ran Liu,
  • Xiaochen Feng,
  • Haoyang Li,
  • Zhong Wang,
  • Xiufeng Zhang,
  • Shangguo Hou

摘要

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.