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Self-interference 3D super-resolution microscopy for deep tissue investigations

Nature Methods volume 15, pages 449–454 (2018)Cite this article

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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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Fig. 1: Determination of the 3D localization of a single emitter by quantitative fluorescence intensity and equiphase imaging.
Fig. 2: Experimental localization precision and accuracy for the SELFI approach compared with that for PSF-shaping with a cylindrical lens.
Fig. 3: Super-resolution imaging of F-actin in fixed adherent cells.
Fig. 4: Super-resolution imaging of F-actin in fixed thick samples.
Fig. 5: Super-resolution imaging of OCT4 in fixed inhomogeneous thick samples.

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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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Authors and Affiliations

  1. Université de Bordeaux, Laboratoire Photonique Numérique et Nanosciences, UMR 5298, F-33400, Talence, France

    Pierre Bon, Jeanne Linarès-Loyez, Maxime Feyeux, Kevin Alessandri, Brahim Lounis, Pierre Nassoy & Laurent Cognet

  2. CNRS and Institut d’Optique, LP2N UMR 5298, F-33400, Talence, France

    Pierre Bon, Jeanne Linarès-Loyez, Maxime Feyeux, Kevin Alessandri, Brahim Lounis, Pierre Nassoy & Laurent Cognet

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Contributions

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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Correspondence to Pierre Bon or Laurent Cognet.

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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).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Notes 1–3

Reporting Summary

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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