Localization of single molecules with structured illumination and structured detection

Luciano A. Masullo

doi:10.1038/s41377-025-01980-1

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Localization of single molecules with structured illumination and structured detection

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- Localization of single molecules with structured illumination and structured detection
- Light: Science & Applications Vol. 14, Issue 11, Pages: 3335-3337(2025)
- Affiliations：
  
  Max Planck Institute of Biochemistry, Planegg 81252, Germany
- Author bio：
  
  Luciano A. Masullo (masullo@biochem.mpg.de)
- Funds：
- DOI：10.1038/s41377-025-01980-1
  CLC：
- Published Online：29 September 2025，
  
  Published：30 November 2025
- Accepted：
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Masullo, L. A. Localization of single molecules with structured illumination and structured detection. Light: Science & Applications, 14, 3335-3337 (2025).
DOI：

Masullo, L. A. Localization of single molecules with structured illumination and structured detection. Light: Science & Applications, 14, 3335-3337 (2025). DOI： 10.1038/s41377-025-01980-1.

摘要

Abstract

A new high-precision single-molecule localization scheme

ISM-FLUX

is an implementation of MINFLUX using image-scanning microscopy (ISM) with a single-photon avalanche diode (SPAD) array detector. ISM-FLUX results in a larger localization range

enhancing the robustness of the localization scheme and it also potentially enables experiments in which absorption and emission of a single fluorophore can be probed independently.

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Keywords

references

Vale, R. D. et al. Direct observation of single kinesin molecules moving along microtubules. Nature 380 , 451–453 (1996)..

Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300 , 2061–2065 (2003)..

Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313 , 1642–1645 (2006)..

Rust, M. J., Bates, M. & Zhuang, X. W. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3 , 793–796 (2006)..

Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl. Acad. Sci. USA 103 , 18911–18916 (2016)..

Enderlein, J. Tracking of fluorescent molecules diffusing within membranes. Appl. Phys. B 71 , 773–777 (2000)..

Ragan, T. et al. 3D particle tracking on a two-photon microscope. J. Fluores . 16 , 325–336 (2006)..

Germann, J. A. & Davis, L. M. Three-dimensional tracking of a single fluorescent nanoparticle using four-focus excitation in a confocal microscope. Opt. Express 22 , 5641–5650 (2014)..

Masullo, L. A., Lopez, L. F. & Stefani, F. D. A common framework for single-molecule localization using sequential structured illumination. Biophys. Rep . 2 , 100036 (2022)..

Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355 , 606–612 (2017)..

Radmacher, N. et al. Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy. Nat. Photonics 18 , 1059–1066 (2024)..

Slenders, E. et al. Array detection enables large localization range for simple and robust MINFLUX. Light. Sci. Appl . 14 , 234 (2025)..

Slenders, E. & Vicidomini, G. ISM-FLUX: MINFLUX with an array detector. Phys. Rev. Res . 5 , 023033 (2023)..

Masullo, L. A. et al. An alternative to MINFLUX that enables nanometer resolution in a confocal microscope. Light. Sci. Appl . 11 , 199 (2022)..

Steen, P. R. et al. The DNA-PAINT palette: a comprehensive performance analysis of fluorescent dyes. Nat. Methods 21 , 1755–1762 (2024)..

Masullo, L. A. et al. Spatial and stoichiometric in situ analysis of biomolecular oligomerization at single-protein resolution. Nat. Commun . 16 , 4202 (2025)..

Reinhardt, S. C. M. et al. Ångström-resolution fluorescence microscopy. Nature 617 , 711–716 (2023)..

Masullo, L. A. et al. Ångström-resolution imaging of cell-surface glycans. Nat. Nanotechnol . https://doi.org/10.1038/s41565-025-01966-5 (2025).

Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17 , 217–224 (2020)..

Deguchi, T. et al. Direct observation of motor protein stepping in living cells using MINFLUX. Science 379 , 1010–1015 (2023)..

Sau, A. et al. Overlapping nuclear import and export paths unveiled by two-colour MINFLUX. Nature 640 , 821–827 (2025)..

Zunino, A. et al. Open-source tools enable accessible and advanced image scanning microscopy data analysis. Nat. Photonics 17 , 457–458 (2023)..

Bucci, A. et al. 4D single-particle tracking with asynchronous read-out single-photon avalanche diode array detector. Nat. Commun . 15 , 6188 (2024)..

Patil, S., Vicidomini, G. & Slenders, E. Open-source 3D active sample stabilization for fluorescence microscopy. Biophys. Rep . 5 , 100208 (2025)..

Masullo, L. A. et al. Pulsed Interleaved MINFLUX. Nano Lett . 21 , 840–846 (2021)..

Lopez, L. F. et al. Optimization and characterization of toroidal foci for super-resolution fluorescence microscopy: tutorial. J. Opt. Soc. Am . 40 , C103–C110 (2023)..

Lopez, L. et al. Open-sour ce sub-nanometer stabilization system for super-resolution fluorescence microscopy. Res. Sq . https://doi.org/10.21203/rs.3.rs-6131181/v1 (2025).

Caprile, F., Masullo, L. A. & Stefani, F. D. PyFocus-a python package for vectorial calculations of focused optical fields under realistic conditions. Application to toroidal foci. Comput. Phys. Commun . 275 , 108315 (2022)..

Marin, Z. & Ries, J. Evaluating MINFLUX experimental performance in silico. Preprint at https://doi.org/10.1101/2025.04.08.647786 https://doi.org/10.1101/2025.04.08.647786 (2025).

Aggarwal, A. V. et al. Fluctuating exciton localization in giant π -conjugated spoked-wheel macrocycles. Nat. Chem . 5 , 964–970 (2013)..

Hedley, G. J. et al. Picosecond time-resolved photon antibunching measures nanoscale exciton motion and the true number of chromophores. Nat. Commun . 12 , 1327 (2021)..

Hildner, R. et al. Quantum coherent energy transfer over varying pathways in single light-harvesting complexes. Science 340 , 1448–1451 (2013)..

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