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Single-molecule orientation and localization microscopy

Nature Photonics volume 19, pages 925–937 (2025)Cite this article

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Abstract

Single-molecule localization microscopy (SMLM) offers enhanced spatial resolution in optical microscopy, providing detailed insights into the spatial organization of proteins in cells at the nanoscale. Over the past decade, SMLM has progressively incorporated the capability to retrieve the orientations of single molecules using their polarized dipolar emission pattern. Here we explore recent advancements in single-molecule orientation and localization microscopy (SMOLM), which yields super-resolved images of molecular three-dimensional (3D) orientations, wobble and 3D positions. This advancement opens possibilities to explore the nanoscale organization and conformation of biological molecules as well as to monitor and design local 3D optical fields in nanophotonics. We cover the principles of SMOLM, discuss recent advances and applications in biology and photonics, and finally highlight exciting future directions and challenges in the field.

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Fig. 1: Principles of SMOLM.
Fig. 2: Measurement and performance assessment in SMOLM.
Fig. 3: Biological applications of SMOLM.
Fig. 4: SMOLM in photonics.

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Acknowledgements

We acknowledge recent contributions to the field by and fruitful discussions with M. A. Alonso, L. A. Aleman-Castaneda, Y. Chen, C. Kumar, I. Herrera, M. Sison, B. Sun and Y. Qiu. This work was funded by Agence Nationale de la Recherche (ANR-20-CE42-0003, ANR-21-CE24-0014, France 2030 Investment Plan IDEC ANR-21-ESRE-0002, Investissements d’Avenir CENTURI ANR-16-CONV-0001, France BioImaging National Infrastructure ANR-10-INBS-04 to S.B.) and the National Institutes of Health (R35GM124858 to M.D.L.).

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  1. Aix Marseille Univ, CNRS, Centrale Med, Institut Fresnel, Marseille, France

    Sophie Brasselet

  2. Preston M. Green Department of Electrical and Systems Engineering, Washington University in St Louis, St Louis, MO, USA

    Matthew D. Lew

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  1. Sophie Brasselet
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Correspondence to Sophie Brasselet or Matthew D. Lew.

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The pixOL microscope mentioned in this work was invented by Tingting Wu and M.D.L. and is covered by US patent 11994470 B2 (2024), which was filed by and assigned to Washington University in St Louis. The raMVR microscope mentioned in this work was invented by Oumeng Zhang and M.D.L., and Washington University has filed a patent application covering the technology (PCT/US2021/063071). The duo-spot microscope mentioned in this work was invented by Tingting Wu, Tianben Ding and M.D.L., and Washington University has filed a patent application covering the technology (PCT/US2021/018235). The tri-spot microscope mentioned in this work was invented by Oumeng Zhang and M.D.L. and is covered by US patent 10761419 B2 (2020), which was filed by and assigned to Washington University in St Louis.

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Brasselet, S., Lew, M.D. Single-molecule orientation and localization microscopy. Nat. Photon. 19, 925–937 (2025). https://doi.org/10.1038/s41566-025-01724-y

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