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Research Article

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Gleb Shtengel, James A. Galbraith, Catherine G. Galbraith, Jennifer Lippincott-Schwartz, Jennifer M. Gillette, Suliana Manley, Rachid Sougrat, Clare M. Waterman, Pakorn Kanchanawong, Michael W. Davidson, Richard D. Fetter, and Harald F. Hess
  1. aHoward Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147;
  2. bNational Institute of Neurological Disorders and Stroke;
  3. cNational Institute of Dental and Craniofacial Research;
  4. dCell Biology and Metabolism Branch, National Institute of Child Health and Human Development; and
  5. eLaboratory of Cell and Tissue Morphodynamics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and
  6. fNational High Magnetic Field Laboratory and Department of Biological Sciences, Florida State University, Tallahassee, FL 32310

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PNAS March 3, 2009 106 (9) 3125-3130; https://doi.org/10.1073/pnas.0813131106
Gleb Shtengel
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James A. Galbraith
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Catherine G. Galbraith
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Jennifer Lippincott-Schwartz
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  • For correspondence: hessh@janelia.hhmi.org jlippin@helix.nih.gov
Jennifer M. Gillette
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Suliana Manley
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Rachid Sougrat
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Clare M. Waterman
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Pakorn Kanchanawong
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Michael W. Davidson
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Richard D. Fetter
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Harald F. Hess
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  • For correspondence: hessh@janelia.hhmi.org jlippin@helix.nih.gov
  1. Contributed by Jennifer Lippincott-Schwartz, December 25, 2008 (received for review November 19, 2008)

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Abstract

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

Keywords:
  • fluorescence microscopy
  • interferometry
  • PALM
  • photoactivated localization microscopy
  • single molecule imaging

Footnotes

  • 1To whom correspondence may be addressed. E-mail: hessh{at}janelia.hhmi.org or jlippin{at}helix.nih.gov
  • Author contributions: G.S., J.A.G., C.G.G., J.L.-S., J.M.G., S.M., R.S., C.M.W., P.K., and H.F.H. designed research; G.S., J.A.G., C.G.G., J.M.G., S.M., R.S., P.K., R.D.F., and H.F.H. performed research; G.S., J.A.G., C.G.G., J.M.G., S.M., P.K., M.W.D., and H.F.H. contributed new reagents/analytic tools; G.S., J.A.G., C.G.G., J.M.G., S.M., P.K., and H.F.H. analyzed data; and G.S., J.A.G., C.G.G., J.L.-S., J.M.G., S.M., C.M.W., P.K., M.W.D., R.D.F., and H.F.H. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0813131106/DCSupplemental.

  • Freely available online through the PNAS open access option.

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Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure
Gleb Shtengel, James A. Galbraith, Catherine G. Galbraith, Jennifer Lippincott-Schwartz, Jennifer M. Gillette, Suliana Manley, Rachid Sougrat, Clare M. Waterman, Pakorn Kanchanawong, Michael W. Davidson, Richard D. Fetter, Harald F. Hess
Proceedings of the National Academy of Sciences Mar 2009, 106 (9) 3125-3130; DOI: 10.1073/pnas.0813131106

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Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure
Gleb Shtengel, James A. Galbraith, Catherine G. Galbraith, Jennifer Lippincott-Schwartz, Jennifer M. Gillette, Suliana Manley, Rachid Sougrat, Clare M. Waterman, Pakorn Kanchanawong, Michael W. Davidson, Richard D. Fetter, Harald F. Hess
Proceedings of the National Academy of Sciences Mar 2009, 106 (9) 3125-3130; DOI: 10.1073/pnas.0813131106
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