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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Datasets generated and/or analyzed in this study are available from J.B. (the corresponding author) upon request.
The data collection and analysis software is available on https://github.com/bewersdorflab/isoSTED, or from J.B. (the corresponding author) upon request.
Abbe, E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Arch. für Mikroskopische Anat. 9, 413–468 (1873).
Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).
Hell, S. & Stelzer, E. H. K. Properties of a 4Pi confocal fluorescence microscope. J. Opt. Soc. Am. A. 9, 2159–2166 (1992).
Bewersdorf, J., Schmidt, R. & Hell, S. W. Comparison of I5M and 4Pi-microscopy. J. Microsc. 222, 105–117 (2006).
Gugel, H. et al. Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy. Biophys. J. 87, 4146–4152 (2004).
Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).
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).
Böhm, U., Hell, S. W. & Schmidt, R. 4Pi-RESOLFT nanoscopy. Nat. Commun. 7, 10504 (2016).
Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. USA 106, 3125–3130 (2009).
Aquino, D. et al. Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat. Methods 8, 353–359 (2011).
Huang, F. et al. Ultra-high resolution 3D imaging of whole cells. Cell 166, 1–13 (2016).
Curdt, F. et al. isoSTED nanoscopy with intrinsic beam alignment. Opt. Express 23, 30891–30903 (2015).
Blom, H. & Widengren, J. Stimulated emission depletion microscopy. Chem. Rev. 117, 7377–7427 (2017).
Lenz, M. O. et al. 3-D stimulated emission depletion microscopy with programmable aberration correction. J. Biophotonics 7, 29–36 (2014).
Booth, M. J. Adaptive optical microscopy: the ongoing quest for a perfect image. Light-Sci. Appl. 3, e165 (2014).
Hao, X., Antonello, J., Allgeyer, E. S., Bewersdorf, J. & Booth, M. J. Aberrations in 4Pi microscopy. Opt. Express 25, 14049–14058 (2017).
Hao, X., Allgeyer, E. S., Booth, M. J. & Bewersdorf, J. Point-spread function optimization in isoSTED nanoscopy. Opt. Lett. 40, 3627–3630 (2015).
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).
Baddeley, D., Carl, C. & Cremer, C. 4Pi microscopy deconvolution with a variable point-spread function. Appl. Opt. 45, 7056–7064 (2006).
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).
Patton, B. R. et al. Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics. Opt. Express 24, 8862–8876 (2016).
Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010).
Gao, M. et al. Expansion stimulated emission depletion microscopy (ExSTED). ACS Nano 12, 4178–4185 (2018).
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).
Gao, R. et al. Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Science 363, eaau8302 (2019).
Bottanelli, F. et al. Two-colour live cell nanoscale imaging of intracellular targets. Nat. Commun. 7, 10778 (2016).
Kilian, N. et al. Assessing photodamage in live-cell STED microscopy. Nat. Methods 15, 755–756 (2018).
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).
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).
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).
Antonello, J. et al. Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy. J. Opt. Soc. Am. A. 31, 1337–1347 (2014).
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).
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).
Robinson, D. N., Cant, K. & Cooley, L. Morphogenesis of Drosophila ovarian ring canals. Development 120, 2015–2025 (1994).
Descloux, A. C., Grussmayer, K. S. & Radenovic, A. Parameter-free image resolution estimation based on decorrelation analysis. Nat. Methods 16, 918–924 (2019).
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.
J.B. discloses a significant financial interest in Bruker Corp. and Hamamatsu Photonics.
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.
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)).
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.
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.
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.
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 Figs. 1–4 and Table 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).
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).
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).
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).
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).
About this article
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