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Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples

Nature Methods volume 5, pages 527–529 (2008)Cite this article

Abstract

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 × 30 × 75nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

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Figure 1: BP FPALM setup and axial resolution.
Figure 2: 3D BP FPALM of 4 μm diameter beads labeled with caged fluorescein.

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Acknowledgements

We thank L. Rocco Carpenter for purification and antibody-conjugation of the caged fluorescein, Th. Tripp for machining services, E. Burns and B. Knowles for valuable comments on the manuscript and J. Spatz for support. This work was supported by the US National Institute of Allergy and Infectious Diseases (K25-65459), the US National Cancer Institute (P30 CA034196), the US National Science Foundation (CHE-0722759 and EPS-0132384), the W.M. Keck Foundation, and start-up funds from The Jackson Laboratory and the University of Maine.

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Authors and Affiliations

  1. Institute for Molecular Biophysics, The Jackson Laboratory, 600 Main Street, Bar Harbor, 04609, Maine, USA

    Manuel F Juette, Mark D Lessard, Michael J Mlodzianoski, Bhupendra S Nagpure & Joerg Bewersdorf

  2. Department of Biophysical Chemistry, and Department of New Materials and Biosystems, University of Heidelberg, Max Planck Institute for Metals Research, Heisenbergstr. 3, Stuttgart, 70569, Germany

    Manuel F Juette

  3. Department of Physics and Astronomy, University of Maine, 120 Bennett Hall, Orono, 04469, Maine, USA

    Travis J Gould, Bhupendra S Nagpure & Samuel T Hess

  4. Institute for Molecular Biophysics, University of Maine, 5737 Jenness Hall, Orono, 04469, Maine, USA

    Travis J Gould & Samuel T Hess

  5. Active Motif, Inc., 1914 Palomar Oaks Way, Carlsbad, 92008, California, USA

    Brian T Bennett

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  1. Manuel F Juette
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Correspondence to Joerg Bewersdorf.

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Competing interests

M.F.J., T.J.G., S.T.H. and J.B. have filed a US patent application covering the work described in this manuscript.

B.T.B. is employed by a commercial company, Active Motif, Inc.

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Supplementary Figures 1–3, Supplementary Methods (PDF 416 kb)

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Juette, M., Gould, T., Lessard, M. et al. Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5, 527–529 (2008). https://doi.org/10.1038/nmeth.1211

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