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<p>An ideal tool for the study of cellular biology would enable the measure of molecular activity nondestructively within living cells. Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells but have historically been limited by a necessary tradeoff between spatiotemporal resolution and throughput. Here we address these limitations using oblique line scan (OLS), a robust single-objective light-sheet-based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a large field of view. We show that OLS can be used to capture protein motion up to 14 μm<sup>2</sup> s<sup>−1</sup> in living cells. We further extend the utility of OLS with in-solution SMT for single-molecule measurement of ligand–protein interactions and disruption of protein–protein interactions using purified proteins. We illustrate the versatility of OLS by showcasing two-color SMT, STORM and single-molecule fluorescence recovery after photobleaching. OLS paves the way for robust, high-throughput, single-molecule investigations of protein function required for basic research, drug screening and systems biology studies.</p>

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Oblique line scan illumination enables expansive, accurate and sensitive single-protein measurements in solution and in living cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年2月18日

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  • Amine Driouchi,
  • Mason Bretan,
  • Brynmor J. Davis,
  • Alec Heckert,
  • Markus Seeger,
  • Maité Bradley Silva,
  • William S. R. Forrest,
  • Jessica Hsiung,
  • Jiongyi Tan,
  • Hongli Yang,
  • David T. McSwiggen,
  • Linda Song,
  • Askhay Sule,
  • Behnam Abaie,
  • Hanzhe Chen,
  • Bryant Chhun,
  • Brianna Conroy,
  • Liam A. Elliott,
  • Eric Gonzalez,
  • Fedor Ilkov,
  • Joshua Isaacs,
  • George Labaria,
  • Michelle Lagana,
  • DeLaine D. Larsen,
  • Brian Margolin,
  • Mai K. Nguyen,
  • Eugene Park,
  • Jeremy Rine,
  • Yangzhong Tang,
  • Martin Vana,
  • Andrew Wilkey,
  • Zhengjian Zhang,
  • Stephen Basham,
  • Jaclyn J. Ho,
  • Stephanie Johnson,
  • Aaron A. Klammer,
  • Kevin Lin,
  • Xavier Darzacq,
  • Eric Betzig,
  • Russell T. Berman,
  • Daniel J. Anderson

摘要

An ideal tool for the study of cellular biology would enable the measure of molecular activity nondestructively within living cells. Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells but have historically been limited by a necessary tradeoff between spatiotemporal resolution and throughput. Here we address these limitations using oblique line scan (OLS), a robust single-objective light-sheet-based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a large field of view. We show that OLS can be used to capture protein motion up to 14 μm2 s−1 in living cells. We further extend the utility of OLS with in-solution SMT for single-molecule measurement of ligand–protein interactions and disruption of protein–protein interactions using purified proteins. We illustrate the versatility of OLS by showcasing two-color SMT, STORM and single-molecule fluorescence recovery after photobleaching. OLS paves the way for robust, high-throughput, single-molecule investigations of protein function required for basic research, drug screening and systems biology studies.