Single-molecule localization and diffusivity microscopy reveals dynamic biomolecular organization in living cells

  • Nat Methods. 2026 May;23(5):1011-1023. doi: 10.1038/s41592-026-03078-x.
Zuhui Wang  #  1  2 ,  Yiwen Liu  #  1  3 ,  Bo Wang  1  2  4 ,  Xiangyu Liu  5 ,  Wulan Deng  6  7  8  9  10  11
Affiliations
  • 1. State Key Laboratory for Gene Function and Modulation Research, Biomedical Pioneering Innovation Center (BIOPIC), Peking University, Beijing, China.
  • 2. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
  • 3. School of Life Sciences, Peking University, Beijing, China.
  • 4. Peking-Tsinghua Center for Life Sciences (CLS), Peking University, Beijing, China.
  • 5. State Key Laboratory of Membrane Biology, Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Pharmaceutical Sciences, Tsinghua University, Beijing, China.
  • 6. State Key Laboratory for Gene Function and Modulation Research, Biomedical Pioneering Innovation Center (BIOPIC), Peking University, Beijing, China. [email protected].
  • 7. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. [email protected].
  • 8. School of Life Sciences, Peking University, Beijing, China. [email protected].
  • 9. Peking-Tsinghua Center for Life Sciences (CLS), Peking University, Beijing, China. [email protected].
  • 10. Beijing Advanced Innovation Center of Genomics, Peking University, Beijing, China. [email protected].
  • 11. State Key Laboratory for Gene Function and Modulation Research, Peking University, Beijing, China. [email protected].
  • # Contributed equally.
PMID: 42050111 DOI: 10.1038/s41592-026-03078-x
Abstract

Single-molecule tracking in living cells measures protein diffusivity but requires sparse imaging, limiting high-density mapping. Here we introduce single-molecule localization and diffusivity microscopy (SMLDM), a deep learning-based approach that accurately estimates single-molecule movement tracks and diffusion coefficients directly from single-frame snapshots, eliminating the need for trajectory linking. Implemented as mobility photoactivated localization microscopy (MPALM) with bright photoactivatable fluorophores and U-Net-based single-molecule segmentation, this method achieves a 50- to 300-fold increase in data density compared to conventional tracking-based approaches, generating high-density, spatially super-resolved maps of molecular diffusivity and organization in living human cells. We applied MPALM to diverse dynamic cellular processes, uncovering nucleosome clustering into low-mobility chromatin domains, pathway-biased μ-opioid receptor dynamic clustering, focal adhesion movement and nonuniform molecular diffusivity and microcondensate organization during early droplet coalescence. SMLDM provides a powerful tool for resolving biomolecular organization and dynamics at single-molecule resolution in live cells.

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