Abstract
Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native cellular processes in intact tissues.
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References
Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).
Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).
Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010).
Kanchanawong, P. et al. Nanoscale architecture of integrin-based cell adhesions. Nature 468, 580–584 (2010).
Rossier, O. et al. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat. Cell Biol. 14, 1057–1067 (2012).
Winckler, P. et al. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells. Sci. Rep. 3, 2387 (2013).
Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).
Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy. Science 347, 543–548 (2015).
Richardson, D. S. & Lichtman, J. W. Clarifying tissue clearing. Cell 162, 246–257 (2015).
Ji, N. Adaptive optical fluorescence microscopy. Nat. Methods 14, 374–380 (2017).
Franke, C., Sauer, M. & van de Linde, S. Photometry unlocks 3D information from 2D localization microscopy data. Nat. Methods 14, 41–44 (2017).
Bourg, N. et al. Direct optical nanoscopy with axially localized detection. Nat. Photonics 9, 587–593 (2015).
Rosen, J. & Brooker, G. Non-scanning motionless fluorescence three-dimensional holographic microscopy. Nat. Photonics 2, 190–195 (2008).
Ram, S., Prabhat, P., Chao, J., Ward, E. S. & Ober, R. J. High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells. Biophys. J. 95, 6025–6043 (2008).
Abrahamsson, S. et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat. Methods 10, 60–63 (2013).
Geissbuehler, S. et al. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging. Nat. Commun. 5, 5830 (2014).
Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. USA 106, 3125–3130 (2009).
Kao, H. P. & Verkman, A. S. Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys. J. 67, 1291–1300 (1994).
Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).
Pavani, S. R. P. & Piestun, R. Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system. Opt. Express 16, 22048–22057 (2008).
Jia, S., Vaughan, J. C. & Zhuang, X. Isotropic 3D super-resolution imaging with a self-bending point spread function. Nat. Photonics 8, 302–306 (2014).
Roider, C., Jesacher, A., Bernet, S. & Ritsch-Marte, M. Axial super-localisation using rotating point spread functions shaped by polarisation-dependent phase modulation. Opt. Express 22, 4029–4037 (2014).
Backer, A. S., Backlund, M. P., von Diezmann, A. R., Sahl, S. J. & Moerner, W. E. A bisected pupil for studying single-molecule orientational dynamics and its application to three-dimensional super-resolution microscopy. Appl. Phys. Lett. 104, 193701 (2014).
Shechtman, Y., Weiss, L. E., Backer, A. S., Lee, M. Y. & Moerner, W. E. Multicolour localization microscopy by point-spread-function engineering. Nat. Photonics 10, 590–594 (2016).
McGorty, R., Schnitzbauer, J., Zhang, W. & Huang, B. Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy. Opt. Lett. 39, 275–278 (2014).
Cuche, E., Marquet, P. & Depeursinge, C. Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms. Appl. Opt. 38, 6994–7001 (1999).
Suck, S. Y., Collin, S., Bardou, N., De Wilde, Y. & Tessier, G. Imaging the three-dimensional scattering pattern of plasmonic nanodisk chains by digital heterodyne holography. Opt. Lett. 36, 849–851 (2011).
Piliarik, M. & Sandoghdar, V. Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites. Nat. Commun. 5, 4495 (2014).
Allier, C. et al. Imaging of dense cell cultures by multiwavelength lens-free video microscopy. Cytometry A 91, 433–442 (2017).
Ober, R. J., Ram, S. & Ward, E. S. Localization accuracy in single-molecule microscopy. Biophys. J. 86, 1185–1200 (2004).
Badieirostami, M., Lew, M. D., Thompson, M. A. & Moerner, W. E. Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane. Appl. Phys. Lett. 97, 161103 (2010).
Booth, M. J., Neil, M. A. A. & Wilson, T. Aberration correction for confocal imaging in refractive-index-mismatched media. J. Microsc. 192, 90–98 (1998).
van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat. Protoc. 6, 991–1009 (2011).
Bon, P., Maucort, G., Wattellier, B. & Monneret, S. Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells. Opt. Express 17, 13080–13094 (2009).
Bon, P., Monneret, S. & Wattellier, B. Noniterative boundary-artifact-free wavefront reconstruction from its derivatives. Appl. Opt. 51, 5698–5704 (2012).
Nieuwenhuizen, R. P. et al. Measuring image resolution in optical nanoscopy. Nat. Methods 10, 557–562 (2013).
Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).
McCauley, H. A. & Wells, J. M. Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish. Development 144, 958–962 (2017).
Cella Zanacchi, F. et al. Live-cell 3D super-resolution imaging in thick biological samples. Nat. Methods 8, 1047–1049 (2011).
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).
Legant, W. R. et al. High-density three-dimensional localization microscopy across large volumes. Nat. Methods 13, 359–365 (2016).
Bon, P. et al. Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy. Nat. Commun. 6, 7764 (2015).
Bon, P., Lécart, S., Fort, E. & Lévêque-Fort, S. Fast label-free cytoskeletal network imaging in living mammalian cells. Biophys. J. 106, 1588–1595 (2014).
Sage, D. et al. Quantitative evaluation of software packages for single-molecule localization microscopy. Nat. Methods 12, 717–724 (2015).
Alessandri, K. et al. A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human neuronal stem cells (hNSC). Lab Chip 16, 1593–1604 (2016).
Xu, K., Babcock, H. P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat.Methods 9, 185–188 (2012).
Acknowledgements
We thank C. Malrieux for help with tissue preparation, E. Fort and S. Lévêque-Fort for fruitful discussions, and L. Groc and J. Ferreira (Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France) for anti-IgG–Alexa Fluor 647. This work was supported by CNRS (to P.N.), the Agence Nationale de la Recherche (ANR-14-OHRI-0001-01 and ANR-15-CE16-0004-03 to L.C.), IdEx Bordeaux (ANR-10-IDEX-03-02 to L.C. ), the France-BioImaging national infrastructure (ANR-10-INBS-04-01 to L.C. and B.L.) and Conseil Regional Nouvelle-Aquitaine (2015-1R60301-00005204 to L.C.).
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P.B., B.L. and L.C. conceived the study; P.B. designed the optical system; P.B. and L.C. supervised the study; K.A., M.F. and P.N. designed and produced the organoid tissues; P.B. and J.L.-L. prepared the samples for dSTORM and performed experiments and data analysis; and P.B., B.L. and L.C. wrote the manuscript. All authors discussed the data and agreed on the final manuscript.
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Integrated supplementary information
Supplementary Figure 1 Gaussian beam propagation.
A beam (yellow) is propagating near a focalization plane and its wavefront (in black) is changing from converging to diverging. We introduce the notations used in the theory: φ and r 0
Supplementary Figure 2 Diffraction grating properties.
(a) Design of the grating. (b) Far-field diffracted energy; the white value indicated the relative energy of each diffracted order and the red dashed line, the encircled energy.
Supplementary Figure 3 Experimental PSF with a low number of photons and Cramèr–Rao lower bound calculated with background noise.
(a) (first row) Measured interferograms of a 100 nm fluorescent bead (tetraspeck) in-focus (z = 0) or defocused within the depth-of-field (z = ±200, ±400 nm) with approximately 1000 photons per PSF. (second row) Numerical Fourier Transform of the interferograms. (b) CRLB considering a photon background of b = 1000 photons/µm².
Supplementary Figure 4 Super-resolution imaging of F-actin in fixed adherent cells (human fibroblasts).
Localization within ~100 nm are shown in different central planes from z = 0 (right-bottom) to z = 660 nm (upper-left). Data are the same as in Fig. 3.
Supplementary Figure 5 Super-resolution imaging of the transcription factor OCT4 at 25 µm: comparison between SELFI and astigmatic PSF-shaping.
~ 6000 detected molecule for both conditions, detections with at least 1500 photons. (a-d) SELFI based 3D super-resolution. (e-h) Astigmatic based 3D super-resolution. (a,e) Super-resolution image. (b,f) Zoom on (a,e) of the axial distribution within a nucleus. (c,g) Full frame z -localization histogram. (d,h) Same (c,g) but only for the considered nucleus in (b,f).
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Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Notes 1–3
41592_2018_5_MOESM3_ESM.zip
Supplementary Software: Stand-alone software for SELFI 3D super-resolution: SELFI calibration, 3D localizations from SELFI-dSTORM acquisitions and 3D imaging rendering
41592_2018_5_MOESM4_ESM.avi
Supplementary Video 1: Numerical z-stack of F-actin in fixed adherent cells (human fibroblasts). Each frame represents localization accumulated in a z-slice of 10 nm
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Bon, P., Linarès-Loyez, J., Feyeux, M. et al. Self-interference 3D super-resolution microscopy for deep tissue investigations. Nat Methods 15, 449–454 (2018). https://doi.org/10.1038/s41592-018-0005-3
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DOI: https://doi.org/10.1038/s41592-018-0005-3