Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy

Florencia Edorna; Florencia D. Choque; Giovanni Ferrari; Luciano A. Masullo; Piotr Zdańkowski; Guillermo P. Acuna; Philip Tinnefeld; Alan M. Szalai; Lucía F. Lopez; Andrés Zelcer; Fernando D. Stefani

doi:10.1038/s41377-025-02022-6

Your Location：

Home >

Browse articles >

Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy

Original Articles

- Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy
- Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy
- Light: Science & Applications 2025年14卷第12期页码：4110-4121
- Affiliations：
  
  1.Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Argentina
  2.Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina
  3.Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Munich, Germany
  4.Warsaw University of Technology, Institute of Micromechanics and Photonics, Warsaw, Poland
  5.Department of Physics, University of Fribourg, Fribourg, Switzerland
  6.Swiss National Center for Competence in Research (NCCR) Bio-inspired Materials, University of Fribourg, Fribourg, Switzerland
  7.Present address: Max Planck Institute of Biochemistry, Planegg, Germany
- Author bio：
  
  Lucía F. Lopez (lucia.lopez@conicet.gov.ar)
  Andrés. Zelcer (andres.zelcer@cibion.conicet.gov.ar)
- Funds：
- DOI：10.1038/s41377-025-02022-6
  中图分类号：
- 收稿：2025-02-28，
  
  修回：2025-08-18，
  
  录用：2025-08-20，
  
  网络出版：2025-11-20，
  
  纸质出版：2025-12-31
- Accepted：
Scan QR Code
Florencia Edorna, Florencia D. Choque, Giovanni Ferrari, 等. Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy[J]. Light: Science & Applications, 2025,14(12):4110-4121.

Edorna, F. et al. Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy. Light: Science & Applications, 14, 4110-4121 (2025).
Florencia Edorna, Florencia D. Choque, Giovanni Ferrari, 等. Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy[J]. Light: Science & Applications, 2025,14(12):4110-4121. DOI： 10.1038/s41377-025-02022-6.

Edorna, F. et al. Open-source sub-nanometer stabilization system for super-resolution fluorescence microscopy. Light: Science & Applications, 14, 4110-4121 (2025). DOI： 10.1038/s41377-025-02022-6.

摘要

Abstract

Recent advances in fluorescence nanoscopy have pushed resolution to the 1–10 nm range

enabling the direct visualization of individual molecules even in crowded biological environments. Achieving this level of precision requires rigorous sample drift control. Techniques such as MINFLUX and RASTMIN

which rely on keeping the sample fixed within an excitation pattern

demand active drift correction to achieve their theoretical nanometer-scale resolution limits. Here

we present an active stabilization system for super-resolution microscopy that delivers sub-nm precision for hours. Featuring a simple optical design

the system can be added as a separate module to any fluorescence microscope. We also provide an open-source control software including a user-friendly graphical interface readily adaptable to different setups. We demonstrate the adaptability and performance of the stabilization system with p-MINFLUX and RASTMIN measurements performed in two different setups

reaching the theoretical Cramér-Rao Bound and resolving ~10 nm distances within DNA origami structures.

关键词

Keywords

references

Lelek, M. et al. Single-molecule localization microscopy. Nat. Rev. Methods Prim. 1 , 39 (2021)..

Lukinavičius, G. et al. Stimulated emission depletion microscopy. Nat. Rev. Methods Prim. 4 , 56 (2024)..

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

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

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

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

Weber, M. et al. MINSTED fluorescence localization and nanoscopy. Nat. Photonics 15 , 361–366 (2021)..

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

Mulhall, E. M. et al. Direct observation of the conformational states of PIEZO1. Nature 620 , 1117–1125 (2023)..

Liu, S., Hoess, P. & Ries, J. Super-resolution microscopy for structural cell biology. Annu. Rev. Biophys. 51 , 301–326 (2022)..

Wolff, J. O. et al. MINFLUX dissects the unimpeded walking of kinesin-1. Science 379 , 1004–1010 (2023)..

Masullo, L. A. et al. Fluorescence nanoscopy at the sub-10 nm scale. Biophysical Rev. 13 , 1101–1112 (2021)..

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

Carter, A. R. et al. Stabilization of an optical microscope to 0.1 nm in three dimensions. Appl. Opt. 46 , 421–427 (2007)..

Jones, S. A. et al. Fast, three-dimensional super-resolution imaging of live cells. Nat. Methods 8 , 499–505 (2011)..

Lee, S. H. et al. Using fixed fiduciary markers for stage drift correction. Opt. Express 20 , 12177–12183 (2012)..

McGorty, R., Kamiyama, D. & Huang, B. Active microscope stabilization in three dimensions using image correlation. Optical Nanoscopy 2 , 3 (2013)..

Grover, G., Mohrman, W. & Piestun, R. Real-time adaptive drift correction for super-resolution localization microscopy. Opt. Express 23 , 23887–23898 (2015)..

Tafteh, R. et al. Real-time 3D stabilization of a super-resolution microscope using an electrically tunable lens. Opt. Express 24 , 22959–22970 (2016)..

Coelho, S. et al. Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells. Sci. Adv. 6 , eaay8271 (2020)..

Mlodzianoski, M. J. et al. Sample drift correction in 3D fluorescence photoactivation localization microscopy. Opt. Express 19 , 15009–15019 (2011)..

Geisler, C. et al. Drift estimation for single marker switching based imaging schemes. Opt. Express 20 , 7274–7289 (2012)..

Wang, Y. N. et al. Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm. Opt. Express 22 , 15982–15991 (2014)..

Shang, M. T. et al. High-precision 3D drift correction with differential phase contrast images. Opt. Express 29 , 34641–34655 (2021)..

Wester, M. J. et al. Robust, fiducial-free drift correction for super-resolution imaging. Sci. Rep. 11 , 23672 (2021)..

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

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

Hwang, W., Bae, S. & Hohng, S. Autofocusing system based on optical astigmatism analysis of single-molecule images. Opt. Express 20 , 29353–29360 (2012)..

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

Schmidt, R. et al. MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nat. Commun. 12 , 1478 (2021)..

Moosmayer, T. et al. MINFLUX fluorescence nanoscopy in biological tissue. Proc. Natl Acad. Sci. USA 121 , e2422020121 (2024)..

Harris et al. Array programming with NumPy. Nature 585 , 357–362 (2020)..

Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods 17 , 261–272 (2020)..

Antonello, J. et al. Multi-scale sensorless adaptive optics: application to stimulated emission depletion microscopy. Opt. Express 28 , 16749–16763 (2020)..

浏览量

Downloads

CSCD

文章被引用时，请邮件提醒。

Submit

工具集

关联资源

暂无数据