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Near-field fluorescence microscopy

An optical nanotool to study protein organization at the cell membrane

  • Original Article
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NanoBiotechnology

Abstract

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods capable of resolving structures with nanometer-scale resolution in intact or living cells. Although fluorescence microscopy has proven to be an extremely versatile tool in cell biology, its diffraction-limited resolution prevents the investigation of membrane compartmentalization at the nanometer scale. Near-field scanning optical microscopy (NSOM) is a relatively unexplored technique that combines both enhanced spatial resolution of probing microscopes and simultaneous measurement of topographic and optical signals. Because of the very small nearfield excitation volume, background fluorescence from the cytoplasm is effectively reduced, enabling the visualization of nano-scale domains on the cell membrane with single molecule detection sensitivity at physiologically relevant packing densities. In this article we discuss technological aspects concerning the implementation of NSOM for cell membrane studies and illustrate its unique advantages in terms of spatial resolution, background suppression, sensitivity, and surface specificity for the study of protein clustering at the cell membrane. Furthermore, we demonstrate reliable operation under physiological conditions, without compromising resolution or sensitivity, opening the road toward truly live cell imaging with unprecedented detail and accuracy.

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Authors and Affiliations

  1. Department of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, University Medical Center, Nijmegen, The Netherlands

    Alessandra Cambi, Frank de Lange & Carl G. Figdor

  2. Applied Optics Group, Faculty of Science and Technology, MESA Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE, Enschede, The Netherlands

    María F. García-Parajó, Bärbel I. de Bakker, Marjolein Koopman & Niek F. van Hulst

Authors
  1. María F. García-Parajó
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  2. Bärbel I. de Bakker
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  3. Marjolein Koopman
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  4. Alessandra Cambi
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  5. Frank de Lange
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  6. Carl G. Figdor
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  7. Niek F. van Hulst
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Correspondence to María F. García-Parajó.

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García-Parajó, M.F., de Bakker, B.I., Koopman, M. et al. Near-field fluorescence microscopy. Nanobiotechnol 1, 113–120 (2005). https://doi.org/10.1385/NBT:1:1:113

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