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3D active stabilization for single-molecule imaging

Nature Protocols volume 16, pages 497–515 (2021)Cite this article

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Abstract

A key part of any super-resolution technique involves accurately correcting for mechanical motion of the sample and setup during acquisition. If left uncorrected, drift degrades the resolution of the final reconstructed image and can introduce unwanted artifacts. Here, we describe how to implement active stabilization, thereby reducing drift to ~1 nm across all three dimensions. In this protocol, we show how to implement our method on custom and standard microscopy hardware. We detail the construction of a separate illumination and detection path, dedicated exclusively to acquiring the diffraction pattern of fiducials deposited on the imaging slide. We also show how to focus lock and adjust the focus in arbitrary nanometer step size increments. Our real-time focus locking is based on kHz calculations performed using the graphics processing unit. The fast calculations allow for rapid repositioning of the sample, which reduces drift below the photon-limited localization precision. Our approach allows for a single-molecule and/or super-resolution image acquisition free from movement artifacts and eliminates the need for complex algorithms or hardware installations. The method is also useful for long acquisitions which span over hours or days, such as multicolor super resolution. Installation does not require specialist knowledge and can be implemented in standard biological laboratories. The full protocol can be implemented within ~2 weeks.

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Fig. 1: Principle of active stabilization.
Fig. 2: Photos of infrared camera path and simplified optical layout on different microscope frames.
Fig. 3: CAD design showing the infrared LED and stage assembly.
Fig. 4: Overview of the graphical user interface.
Fig. 5: Active stabilization does not exhibit residual drift.
Fig. 6: Side-by-side comparison of raw data of F-actin obtained with active stabilization and without drift correction.

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Data availability

The design for the bracket specific to the RM21 is available from GitHub (https://github.com/spcoelho/Active-Stabilization-Design). Microscope control and the Simulation and Hardware Test software is available from GitHub (https://github.com/spcoelho/Active-Stabilization.git). Example data for Figs. 1 and 5, and Supplementary Figs. 1 and 2 can be found on GitHub (https://github.com/spcoelho/Active-Stabilization-Test-Data) and image data can be found on the Zenodo online repository (https://doi.org/10.5281/zenodo.3973720 and https://doi.org/10.5281/zenodo.3974141).

Code availability

The active stabilization software, including the executables can be found on GitHub: https://github.com/spcoelho/Active-Stabilization.git

References

  1. Hell, S. W. et al. The 2015 super-resolution microscopy roadmap. J. Phys. D. Appl. Phys. 48, 443001 (2015).

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  3. Hess, S. T., Girirajan, T. P. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Nicovich, P. R., Owen, D. M. & Gaus, K. Turning single-molecule localization microscopy into a quantitative bioanalytical tool. Nat. Protoc. 12, 453–460 (2017).

    CAS  PubMed  Google Scholar 

  7. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  9. Gustafsson, M. G. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102, 13081–13086 (2005).

    CAS  PubMed  Google Scholar 

  10. Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    CAS  PubMed  Google Scholar 

  11. Pawley, J. Handbook of Biological Confocal Microscopy Vol. 236 (Springer Science & Business Media, 2006).

  12. Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198 (2017).

    CAS  PubMed  Google Scholar 

  13. Deschout, H. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nat. Methods 11, 253 (2014).

    CAS  PubMed  Google Scholar 

  14. Moerner, W. E. New directions in single-molecule imaging and analysis. Proc. Natl Acad. Sci. USA 104, 12596–12602 (2007).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Huhle, A. et al. Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy. Nat. Commun. 6, 1–8 (2015).

    Google Scholar 

  17. Fish, K. N. Total internal reflection fluorescence (TIRF) microscopy. Curr. Protoc. Cytom. 50, 12.18. 11–12.18. 13 (2009).

    Google Scholar 

  18. Kim, J. et al. Unveiling the relationship between the perovskite precursor solution and the resulting device performance. J. Am. Chem. Soc. 142, 6251–6260 (2020).

    CAS  PubMed  Google Scholar 

  19. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

    CAS  PubMed  Google Scholar 

  20. Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Pavani, S. R. P. et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl Acad. Sci. 106, 2995–2999 (2009).

    CAS  PubMed  Google Scholar 

  22. Shechtman, Y., Sahl, S. J., Backer, A. S. & Moerner, W. Optimal point spread function design for 3D imaging. Phys. Rev. Lett. 113, 133902 (2014).

    PubMed  PubMed Central  Google Scholar 

  23. Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. 106, 3125–3130 (2009).

    CAS  PubMed  Google Scholar 

  24. Shroff, H., Galbraith, C. G., Galbraith, J. A. & Betzig, E. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat. Methods 5, 417 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Diekmann, R. et al. Chip-based wide field-of-view nanoscopy. Nat. Photonics 11, 322–328 (2017).

    CAS  Google Scholar 

  26. Huang, F. et al. Ultra-high resolution 3D imaging of whole cells. Cell 166, 1028–1040 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Gao, L., Shao, L., Chen, B.-C. & Betzig, E. 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat. Protoc. 9, 1083–1101 (2014).

    CAS  PubMed  Google Scholar 

  28. Baek, J. et al. Imaging galectin-3 dependent endocytosis with lattice light-sheet microscopy. SPIE Proceedings Vol. 10340 (eds. Sampson, D. D. et al.) (SPIE, 2017).

  29. Aoki, K. & Matsuda, M. Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nat. Protoc. 4, 1623 (2009).

    CAS  PubMed  Google Scholar 

  30. Poland, S. P. et al. A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging. Biomed. Opt. Express 6, 277–296 (2015).

    PubMed  PubMed Central  Google Scholar 

  31. Poland, S. P. et al. Time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo. Opt. Lett. 39, 6013–6016 (2014).

    CAS  PubMed  Google Scholar 

  32. Suhling, K. et al. Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments. Med. Photonics 27, 3–40 (2015).

    Google Scholar 

  33. Suhling, K. et al. in Handbook of Photonics for Biomedical Engineering 353–405 (Springer, 2017).

  34. Suhling, K. et al. in Advanced Time-Correlated Single Photon Counting Applications 119–188 (Springer, 2015).

  35. Burke, D., Patton, B., Huang, F., Bewersdorf, J. & Booth, M. J. Adaptive optics correction of specimen-induced aberrations in single-molecule switching microscopy. Optica 2, 177–185 (2015).

    CAS  Google Scholar 

  36. Coelho, S. et al. Multifocal multiphoton microscopy with adaptive optical correction. SPIE Proceedings Vol. 8588 (SPIE, 2013).

  37. Coelho, S., Poland, S. P., Devauges, V. & Ameer-Beg, S. M. Adaptive optics for a time-resolved Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) in vivo. Opt. Lett. 45, 2732–2735 (2020).

    PubMed  PubMed Central  Google Scholar 

  38. Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009).

    CAS  PubMed  Google Scholar 

  39. Boutros, M., Heigwer, F. & Laufer, C. Microscopy-based high-content screening. Cell 163, 1314–1325 (2015).

    CAS  PubMed  Google Scholar 

  40. Gustavsson, A.-K., Petrov, P. N., Lee, M. Y., Shechtman, Y. & Moerner, W. 3D single-molecule super-resolution microscopy with a tilted light sheet. Nat. Commun. 9, 1–8 (2018).

    CAS  Google Scholar 

  41. Giessibl, F. J. Advances in atomic force microscopy. Rev. Mod. Phys. 75, 949 (2003).

    CAS  Google Scholar 

  42. Schmidt, P. D., Reichert, B. H., Lajoie, J. G. & Sivasankar, S. Method for high frequency tracking and sub-nm sample stabilization in single molecule fluorescence microscopy. Sci. Rep. 8, 13912 (2018).

    PubMed  PubMed Central  Google Scholar 

  43. Balinovic, A., Albrecht, D. & Endesfelder, U. Spectrally red-shifted fluorescent fiducial markers for optimal drift correction in localization microscopy. J. Phys. D. Appl. Phys. 52, 204002 (2019).

    CAS  Google Scholar 

  44. Henriques, R. et al. QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat. Methods 7, 339–340 (2010).

    CAS  PubMed  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  46. Pertsinidis, A., Zhang, Y. & Chu, S. Subnanometre single-molecule localization, registration and distance measurements. Nature 466, 647–651 (2010).

    CAS  PubMed  Google Scholar 

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

    Google Scholar 

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

    CAS  PubMed  Google Scholar 

  49. Ma, H., Xu, J., Jin, J., Huang, Y. & Liu, Y. A simple marker-assisted 3D nanometer drift correction method for superresolution microscopy. Biophys. J. 112, 2196–2208 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Gosse, C. & Croquette, V. Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys. J. 82, 3314–3329 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. van Loenhout, M. T. J., Kerssemakers, J. W. J., De Vlaminck, I. & Dekker, C. Non-bias-limited tracking of spherical particles, enabling nanometer resolution at low magnification. Biophys. J. 102, 2362–2371 (2012).

    PubMed  PubMed Central  Google Scholar 

  52. Chivers, C. E., Koner, A. L., Lowe, E. D. & Howarth, M. How the biotin–streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer. Biochem. J. 435, 55–63 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Haber, C. & Wirtz, D. Magnetic tweezers for DNA micromanipulation. Rev. Sci. Instrum. 71, 4561–4570 (2000).

    CAS  Google Scholar 

  54. Bormuth, V. et al. Optical trapping of coated microspheres. Opt. Express 16, 13831–13844 (2008).

    CAS  PubMed  Google Scholar 

  55. Huang, B., Jones, S. A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Methods 5, 1047–1052 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. van de Linde, S. Single-molecule localization microscopy analysis with ImageJ. J. Phys. D. Appl. Phys. 52, 203002 (2019).

    Google Scholar 

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Acknowledgements

We are grateful for support from the Australia Research Council (CE140100011 to K.G., FL150100060 and CE140100036 to J.J.G.) and National Health and Medical Research Council of Australia (APP1059278 to K.G.). We also thank L. Lee and Y. Gambin for useful discussions; M. Catarino, M. Farrell, and J. Goyette for assistance with the manuscript preparation; and M. Farrell for help with software troubleshooting.

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Author notes

  1. Jongho Baek

    Present address: NetTargets, National Nanofab Center, KAIST, Daejeon, Republic of Korea

  2. These authors contributed equally: Simao Coelho, Jongho Baek.

Authors and Affiliations

  1. EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia

    Simao Coelho, Jongho Baek, James Walsh & Katharina Gaus

  2. ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia

    Simao Coelho, Jongho Baek, James Walsh & Katharina Gaus

  3. School of Chemistry and Australian Centre of NanoMedicine, University of New South Wales, Sydney, Australia

    J. Justin Gooding

  4. ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of New South Wales, Sydney, Australia

    J. Justin Gooding

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Contributions

S.C. and J.B. designed and implemented the active stabilization, built the original Feedback SMLM, and performed the experiments. S.C. and J.W. implemented active stabilization on the Nikon Ti. J.W. implemented active stabilization custom microscope units. S.C., J.J.G., and K.G. wrote the manuscript.

Corresponding authors

Correspondence to Simao Coelho or Katharina Gaus.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Gerhard Schütz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol Coelho, S. et al. Sci. Adv. 6, eaay8271 (2020): https://advances.sciencemag.org/content/6/16/eaay8271

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Coelho, S., Baek, J., Walsh, J. et al. 3D active stabilization for single-molecule imaging. Nat Protoc 16, 497–515 (2021). https://doi.org/10.1038/s41596-020-00426-9

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