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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

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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.

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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.

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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.

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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

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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.

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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).

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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

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