Oblique line scan illumination enables expansive, accurate and sensitive single-protein measurements in solution and in living cells
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.
Authors
- Jessica Hsiung
- Jaclyn J. Ho (ORCID: https://orcid.org/0000-0001-9166-5953)
- Stephanie L. Johnson (ORCID: https://orcid.org/0000-0002-6198-9364)
- Michelle Lagana
- Alec Heckert (ORCID: https://orcid.org/0000-0001-8748-6645)
- Mason Bretan (ORCID: https://orcid.org/0000-0002-4133-9807)
- Brianna Conroy (ORCID: https://orcid.org/0009-0009-0688-7750)
- George Labaria
- Stephen E. Basham (ORCID: https://orcid.org/0009-0007-1348-4672)
- F. A. Ilkov (ORCID: https://orcid.org/0000-0002-3700-7033)
- Yangzhong Tang (ORCID: https://orcid.org/0000-0001-7086-0063)
- Markus Seeger (ORCID: https://orcid.org/0000-0002-4588-0393)
- Kevin Lin (ORCID: https://orcid.org/0000-0001-7115-9199)
- Eric Betzig (ORCID: https://orcid.org/0000-0002-4192-1274)
- Aaron Klammer (ORCID: https://orcid.org/0000-0002-6796-9188)
- Mai K. Nguyen (ORCID: https://orcid.org/0000-0003-3550-7728)
- Jiongyi Tan
- Bryant B. Chhun (ORCID: https://orcid.org/0000-0002-3539-2396)
- Zhengjian Zhang (ORCID: https://orcid.org/0000-0002-2840-0837)
- Amine Driouchi (ORCID: https://orcid.org/0000-0002-2940-1161)
- DeLaine D. Larsen (ORCID: https://orcid.org/0000-0002-9689-8454)
- Daniel J. Anderson (ORCID: https://orcid.org/0000-0002-3249-2439)
- Joshua Isaacs (ORCID: https://orcid.org/0000-0003-1509-5359)
- Brian Margolin (ORCID: https://orcid.org/0000-0003-3365-7677)
- Xavier Darzacq (ORCID: https://orcid.org/0000-0003-2537-8395)
- Hanzhe Chen (ORCID: https://orcid.org/0000-0001-5727-2224)
- David T. McSwiggen (ORCID: https://orcid.org/0000-0003-3844-7433)
- L. Elliott (ORCID: https://orcid.org/0009-0001-2626-4130)
- Eric Gonzalez (ORCID: https://orcid.org/0000-0002-3500-9668)
- Behnam Abaie
- Jeremy Rine
- Andrew Wilkey (ORCID: https://orcid.org/0009-0003-8328-5389)
- William S. R. Forrest (ORCID: https://orcid.org/0000-0003-0985-1453)
- Eugene Park (ORCID: https://orcid.org/0000-0001-8432-5168)
- Linda Song
- Russell Berman (ORCID: https://orcid.org/0000-0001-8205-5627)
- Martin Vana (ORCID: https://orcid.org/0009-0003-4769-8282)
- Brynmor J. Davis
- Hongli Yang
- Askhay Sule
- Maité Bradley Silva
Institutions
- Estonian Information Technology College (EE)
- University of California, Berkeley (US)
Publication Details
- Journal
- Nature Methods
- Published
- 2025-02-18
- DOI
- https://doi.org/10.1038/s41592-025-02594-6
- Citations
- 12
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 19.61