Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution

Nature Methods volume 18, pages 688–693 (2021)Cite this article

Subjects

Abstract

Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Principle and performance of AO isoSTED nanoscopy.
Fig. 2: Applications of AO isoSTED nanoscopy in cultured mammalian cells.
Fig. 3: IsoSTED images of Drosophila egg chambers and mouse hippocampal brain tissue.

Similar content being viewed by others

Data availability

Datasets generated and/or analyzed in this study are available from J.B. (the corresponding author) upon request.

Code availability

The data collection and analysis software is available on https://github.com/bewersdorflab/isoSTED, or from J.B. (the corresponding author) upon request.

References

  1. Abbe, E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Arch. für Mikroskopische Anat. 9, 413–468 (1873).

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Hell, S. & Stelzer, E. H. K. Properties of a 4Pi confocal fluorescence microscope. J. Opt. Soc. Am. A. 9, 2159–2166 (1992).

    Article  Google Scholar 

  4. Bewersdorf, J., Schmidt, R. & Hell, S. W. Comparison of I5M and 4Pi-microscopy. J. Microsc. 222, 105–117 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Gugel, H. et al. Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy. Biophys. J. 87, 4146–4152 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Dyba, M. & Hell, S. W. Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution. Phys. Rev. Lett. 88, 163901 (2002).

    Article  PubMed  Google Scholar 

  8. Böhm, U., Hell, S. W. & Schmidt, R. 4Pi-RESOLFT nanoscopy. Nat. Commun. 7, 10504 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Aquino, D. et al. Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat. Methods 8, 353–359 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Huang, F. et al. Ultra-high resolution 3D imaging of whole cells. Cell 166, 1–13 (2016).

    Article  Google Scholar 

  12. Curdt, F. et al. isoSTED nanoscopy with intrinsic beam alignment. Opt. Express 23, 30891–30903 (2015).

    Article  PubMed  Google Scholar 

  13. Blom, H. & Widengren, J. Stimulated emission depletion microscopy. Chem. Rev. 117, 7377–7427 (2017).

    Article  CAS  PubMed  Google Scholar 

  14. Lenz, M. O. et al. 3-D stimulated emission depletion microscopy with programmable aberration correction. J. Biophotonics 7, 29–36 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Booth, M. J. Adaptive optical microscopy: the ongoing quest for a perfect image. Light-Sci. Appl. 3, e165 (2014).

    Article  Google Scholar 

  16. Hao, X., Antonello, J., Allgeyer, E. S., Bewersdorf, J. & Booth, M. J. Aberrations in 4Pi microscopy. Opt. Express 25, 14049–14058 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Hao, X., Allgeyer, E. S., Booth, M. J. & Bewersdorf, J. Point-spread function optimization in isoSTED nanoscopy. Opt. Lett. 40, 3627–3630 (2015).

    Article  PubMed  Google Scholar 

  18. Barentine, A. E. S., Schroeder, L. K., Graff, M., Baddeley, D. & Bewersdorf, J. Simultaneously measuring image features and resolution in live-cell STED images. Biophys. J. 115, 951–956 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Baddeley, D., Carl, C. & Cremer, C. 4Pi microscopy deconvolution with a variable point-spread function. Appl. Opt. 45, 7056–7064 (2006).

    Article  PubMed  Google Scholar 

  20. Gould, T. J., Burke, D., Bewersdorf, J. & Booth, M. J. Adaptive optics enables 3D STED microscopy in aberrating specimens. Opt. Express 20, 20998–21009 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Patton, B. R. et al. Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics. Opt. Express 24, 8862–8876 (2016).

    Article  PubMed  Google Scholar 

  22. Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gao, M. et al. Expansion stimulated emission depletion microscopy (ExSTED). ACS Nano 12, 4178–4185 (2018).

    Article  CAS  PubMed  Google Scholar 

  24. Piazza, S., Bianchini, P., Sheppard, C., Diaspro, A. & Duocastella, M. Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping. J. Biophotonics 11, e201870129 (2018).

    Article  Google Scholar 

  25. Gao, R. et al. Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Science 363, eaau8302 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bottanelli, F. et al. Two-colour live cell nanoscale imaging of intracellular targets. Nat. Commun. 7, 10778 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kilian, N. et al. Assessing photodamage in live-cell STED microscopy. Nat. Methods 15, 755–756 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Booth, M., Wilson, T., Sun, H. B., Ota, T. & Kawata, S. Methods for the characterization of deformable membrane mirrors. Appl. Opt. 44, 5131–5139 (2005).

    Article  PubMed  Google Scholar 

  29. Takeda, M., Ina, H. & Kobayashi, S. Fourier-transform method of fringe-pattern analysis for computer-based topography and interferometry. J. Opt. Soc. Am. 72, 156–160 (1982).

    Article  Google Scholar 

  30. Herraez, M. A., Burton, D. R., Lalor, M. J. & Gdeisat, M. A. Fast two-dimensional phase-unwrapping algorithm based on sorting by reliability following a noncontinuous path. Appl. Opt. 41, 7437–7444 (2002).

    Article  PubMed  Google Scholar 

  31. Antonello, J. et al. Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy. J. Opt. Soc. Am. A. 31, 1337–1347 (2014).

    Article  Google Scholar 

  32. Duden, R., Griffiths, G., Frank, R., Argos, P. & Kreis, T. E. β-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to β-adaptin. Cell 64, 649–665 (1991).

    Article  CAS  PubMed  Google Scholar 

  33. Singleton, K. & Woodruff, R. I. The osmolarity of adult Drosophila hemolymph and its effect on oocyte-nurse cell electrical polarity. Dev. Biol. 161, 154–167 (1994).

    Article  CAS  PubMed  Google Scholar 

  34. Robinson, D. N., Cant, K. & Cooley, L. Morphogenesis of Drosophila ovarian ring canals. Development 120, 2015–2025 (1994).

    Article  CAS  PubMed  Google Scholar 

  35. Descloux, A. C., Grussmayer, K. S. & Radenovic, A. Parameter-free image resolution estimation based on decorrelation analysis. Nat. Methods 16, 918–924 (2019).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank G. Sirinakis, now at the University of Cambridge, Y. Zhang, now at the Westlake University, A.E.S. Barentine and M.G. Velasco, now at the Abberior Instruments, and E.B. Kromann, now at the Technical University of Denmark, from Yale University, for sharing the source code and fruitful discussions. We also thank Y. Wang at the University of Utah for technical support with FluoRender. This work was supported by the Wellcome Trust (grant nos. 095927/A/11/Z to J.B. and J.E.R.; 095927/B/11/Z to M.J.B., 203285/B/16/Z to J.E.R.; 203285/C/16/Z to M.J.B.), the G. Harold & Leila Y. Mathers Foundation to J.B., the National Institutes of Health (grant nos. P30 DK045735 to J.B.; R01 GM043301 to L.C.; R01 DA018928 to T.B.) and the European Research Council (AdOMiS, grant no. 695140) to M.J.B.

Author information

Author notes

  1. Xiang Hao

    Present address: State Key Laboratory of Modern Optical Instrumentation, College of Optical Science and Technology, Zhejiang University, Hangzhou, China

  2. Edward S. Allgeyer

    Present address: The Gurdon Institute, University of Cambridge, Cambridge, UK

  3. Lena K. Schroeder

    Present address: Cellular Imaging Shared Resource, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

  4. Francesca Bottanelli

    Present address: Department of Biology, Chemistry and Pharmacy, Free University of Berlin, Berlin, Germany

  5. Jiaxi Zhao

    Present address: Department of Physics, University of California, Berkeley, Berkeley, CA, USA

  6. These authors contributed equally: Xiang Hao, Edward S. Allgeyer, Dong-Ryoung Lee.

Authors and Affiliations

  1. Department of Cell Biology, Yale School of Medicine, New Haven, CT, USA

    Xiang Hao, Edward S. Allgeyer, Dong-Ryoung Lee, Lena K. Schroeder, Francesca Bottanelli, Jiaxi Zhao, Phylicia Kidd, Mark D. Lessard, James E. Rothman, Lynn Cooley & Joerg Bewersdorf

  2. Centre for Neural Circuits and Behaviour, University of Oxford, Oxford, UK

    Jacopo Antonello & Martin J. Booth

  3. Department of Engineering Science, University of Oxford, Oxford, UK

    Jacopo Antonello & Martin J. Booth

  4. Department of Neuroscience, Tufts University School of Medicine, Boston, MA, USA

    Katherine Watters & Thomas Biederer

  5. Department of Neurology, Yale School of Medicine, New Haven, CT, USA

    Katherine Watters & Thomas Biederer

  6. Department of Genetics, Yale University, New Haven, CT, USA

    Julianne A. Gerdes & Lynn Cooley

  7. Nanobiology Institute, Yale University, West Haven, CT, USA

    James E. Rothman & Joerg Bewersdorf

  8. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA

    Lynn Cooley

  9. Department of Biomedical Engineering, Yale University, New Haven, CT, USA

    Joerg Bewersdorf

Authors
  1. Xiang Hao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Edward S. Allgeyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dong-Ryoung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jacopo Antonello
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Katherine Watters
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julianne A. Gerdes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lena K. Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Francesca Bottanelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jiaxi Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Phylicia Kidd
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Mark D. Lessard
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. James E. Rothman
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Lynn Cooley
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Thomas Biederer
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Martin J. Booth
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Joerg Bewersdorf
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.H., E.S.A. and J.B. designed the instrument. X.H., E.S.A., J.A. and D.-R.L. built the instrument and developed the software. X.H., J.A., J.Z. and M.J.B. developed and optimized the AO strategy. X.H., D.-R.L. and E.S.A. optimized and tested the instrument. K.W., J.A.G., L.K.S., F.B., P.K., M.D.L., J.E.R., L.C., T.B. and J.B. designed the biological experiments. K.W., J.A.G., L.K.S., F.B., P.K. and M.D.L. optimized the sample preparation protocols and prepared the samples. X.H., D.-R.L., M.D.L. and J.B. visualized the data. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to Joerg Bewersdorf.

Ethics declarations

Competing interests

J.B. discloses a significant financial interest in Bruker Corp. and Hamamatsu Photonics.

Additional information

Peer review information Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Design of isoSTED nanoscope.

a, System layout. The system is built from the following components: excitation lasers (594 nm, 650 nm), depletion laser (775 nm), acousto-optic modulator (AOM), polarization-maintaining single-mode fibers (curvy orange lines), multi-mode fibers (curvy grey lines), half-wave plate (HWP), quarter-wave plate (QWP), spatial light modulator (SLM), scanning mirror module (including one resonant mirror and two galvanometer mirrors), polarizing beam splitter (PBS), dichroic mirrors, lenses, mirrors, bandpass filters, avalanche photo diodes (APDs), and control electronics. b, CAD rendering of the isoSTED instrument. c, Transmission spectra of the detection bandpass filters and the custom-made quad-bandpass dichroic mirror (highlighted in yellow in (a)).

Extended Data Fig. 2 Quantification of 3D resolution.

Crimson fluorescent beads with 20 nm diameter were imaged in isoSTED mode. One-dimensional Lorentzian functions were fitted to images of isolated beads in both x and z directions. The full-width-half-maximum (FWHM) of the fitted functions are marked above each bead image. The numbers in green and red indicate the corresponding FWHMs in lateral (x) and axial (z) directions, respectively. All images are normalized to their peak intensities.

Extended Data Fig. 3 Effects of refractive index mismatch on the depletion and effective PSF.

The presence of refractive index mismatch between the silicone oil (ns) and the sample mounting medium (ni) causes the appearance of depth dependent side-lobes in the effective PSFs, even when correcting for piston. In addition, the main lobe of the effective PSF also shifts as a function of depth. All results are calculated for ns= 1.406 and ni = 1.33.

Extended Data Fig. 4 Relative fluorescence intensity of ten consecutive 3D data stacks.

a, Sum projections of a representative series of ten 3D data stacks consecutively recorded of the same cell region (N=5 cell regions). A photobleaching curve is extracted for each series. b, Photobleaching as a function of iterations. Data points and error bars represent the mean values of five different data sets and their standard deviations, respectively.

Extended Data Fig. 5 Sample holder design.

The specimens are mounted on a ring-shaped sample holder (a), between two coverslips at 5–10 μm (b, cells) or ~30 μm (c, tissue) distance. 100-nm gold beads are sparsely deposited onto the bottom coverslip for use during the aberration correction.

Extended Data Fig. 6 Magnified Fig. 1e for better visualization.

xz|y=0 cross sections of the effective PSFs as measured with a 20-nm diameter crimson fluorescent bead. From top to bottom: single-objective confocal PSF, isoSTED PSFs with (bottom) and without (middle) defocus added to the STEDz pattern. To the right of each PSF, the respective axial intensity profiles (z) are displayed. The raw data (black dots) are fitted with a Lorentzian function (red curves), and the corresponding FWHMs are highlighted in blue.

Extended Data Fig. 7 Magnified Fig. 3b for better visualization.

Effects of AO on the imaging performance of isoSTED. The left and middle columns show the xy|z = 0 (left) and xz|y = 0 (middle) cross-sections of the PSFs with (bottom two rows) and without (top two rows) AO aberration correction. The PSFs were obtained by imaging 150-nm-diameter gold beads. The right column shows 2D images of ring canals in a Drosophila egg chamber recorded in confocal and isoSTED modes. The F-actin proteins on the ring canals are immunolabeled with ATTO 594.

Extended Data Fig. 8 Magnified Fig. 3i for better visualization.

F-actin staining of six ring canals in the same egg chamber. At the top-right corner of each sub-figure, the ring thickness is given (measured as the FWHM of the intensity profile in radial direction in the corresponding 2D ring).

Extended Data Fig. 9 Magnified Fig. 3m for better visualization.

Magnified projection images of the pre- and postsynaptic labels at the positions highlighted in main text Fig. 3j. The trans-synaptic axes in these figures are rotated into the viewing planes.

Extended Data Fig. 10 isoSTED and confocal images of calbindin in mouse cerebellar sections at different depths (z).

a, xy cross sections of isoSTED and confocal microscopy (CM) images at different depths. b, Resolution at different depths (n = 3 independent experiments). Data points and error bars represent the mean values of three different data sets and the respective standard deviation. An equivalent analysis of the CM images yields an average resolution of 307 nm.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Table 1.

Reporting Summary

Supplementary Video 1

AO isoSTED images of synaptonemal complexes in a mouse spermatocyte. Video rendering of the data shown in Fig. 2a. One-color image of synaptonemal complexes (SYCP1 immunolabeled with ATTO 647N).

Supplementary Video 2

AO isoSTED images of COPI vesicles and Golgi apparatus in a HeLa cell. Video rendering of the data shown in Fig. 2e. Two-color image of COPI vesicles (βCOP immunolabeled with ATTO 647N) and Golgi apparatus (GM130 immunolabeled with ATTO 594).

Supplementary Video 3

AO isoSTED images of endoplasmic reticulum and mitochondria in a COS-7 cell. Video rendering of the data shown in Fig. 2j. Two-color image of endoplasmic reticulum (Sec61β immunolabeled with ATTO 594) and mitochondria (TOM20 immunolabeled with ATTO 647N).

Supplementary Video 4

AO isoSTED images of ring canals in Drosophila egg chambers. Video rendering of the data shown in Fig. 3d. Two-color image of F-actin (immunolabeled with ATTO 594) and HtsRC (immunolabeled with ATTO 647N).

Supplementary Video 5

AO isoSTED images of excitatory synapses in a 30-μm-thick mouse hippocampal brain sections. Video rendering of the data shown in Fig. 3j. Two-color image of presynaptic active zone marker Bassoon (immunolabeled with ATTO 594) and postsynaptic scaffolding molecule Homer1 (immunolabeled with ATTO 647N).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hao, X., Allgeyer, E.S., Lee, DR. et al. Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution. Nat Methods 18, 688–693 (2021). https://doi.org/10.1038/s41592-021-01149-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-021-01149-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing