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Live-cell 3D super-resolution imaging in thick biological samples

Nature Methods volume 8, pages 1047–1049 (2011)Cite this article

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Abstract

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50–150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

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Figure 1: IML-SPIM 3D super-resolution imaging of nanocapsules.
Figure 2: IML-SPIM super-resolution imaging of human mammary MCF10A cell spheroids expressing H2B-PAmCherry.
Figure 3: IML-SPIM imaging of cell spheroids expressing connexin 43–PAmCherry.

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Acknowledgements

We thank E.H.K. Stelzer, P. Keller, T.J. Gould and S.T. Hess for software and experimental training and helpful discussions, C.J.R. Sheppard, K. Braeckmans, I. Testa, G. Vicidomini, D. Mazza, S. Galiani, E. Ronzitti, B. Harke, P. Bianchini, V. Murino and R. Cingolani for critical discussions and A. Giampaoli for help editing the text. PAmCherry fusion protein was a gift from V.V. Verkhusha (Albert Einstein College of Medicine). Work partially funded by Italian Foundation for Cancer Research Institute of Molecular Oncology, Milan, Italy), EU FP7 project Single or few molecules detection by combined enhanced spectroscopies GA 229375 and Italian Programmi di ricerca di rilevante interesse nazionale 2008JZ4MLB grants.

Author information

Authors and Affiliations

  1. Istituto Italiano di Tecnologia, Genova, Italia

    Francesca Cella Zanacchi, Zeno Lavagnino, Michela Perrone Donnorso, Alessio Del Bue & Alberto Diaspro

  2. MicroScoBio Dipartimento di Fisica Università di Genova, Istituto Fondazione italiana ricerca sul cancro di Oncologia Molecolare, Genova, Italia

    Zeno Lavagnino & Alberto Diaspro

  3. Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

    Laura Furia & Mario Faretta

Authors
  1. Francesca Cella Zanacchi
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  2. Zeno Lavagnino
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  3. Michela Perrone Donnorso
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  4. Alessio Del Bue
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  5. Laura Furia
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  6. Mario Faretta
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  7. Alberto Diaspro
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Contributions

F.C.Z. and A.D. conceived the IML-SPIM imaging concept, conceived the study, designed experiments and wrote the manuscript. F.C.Z. and Z.L. realized the optical set-up and data acquisition. F.C.Z. realized imaging and data analysis. M.P.D. and F.C.Z. realized polyelectrolyte nanocapsules. M.F. and L.F. prepared biological samples. A.D.B. wrote the software tool for 3D analysis. F.C.Z., Z.L., M.F. and A.D. refined the manuscript. A.D. supervised the project.

Corresponding author

Correspondence to Alberto Diaspro.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9, Supplementary Results 1–4 (PDF 1740 kb)

Supplementary Video 1

Axial optical sectioning in a spheroid using IML-SPIM. IML-SPIM provides three-dimensional super-resolution images of nuclei in human mammary MCF10A cell spheroids expressing H2B-PAmCherry (experimental details are provided in Supplementary Fig. 6). The movie steps through x-y slices with 116 nm z separation. Scale bar, 10 μm. (AVI 277 kb)

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Cella Zanacchi, F., Lavagnino, Z., Perrone Donnorso, M. et al. Live-cell 3D super-resolution imaging in thick biological samples. Nat Methods 8, 1047–1049 (2011). https://doi.org/10.1038/nmeth.1744

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