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Biophys. J. BioFAST: First Published February 4, 2005. doi:10.1529/biophysj.104.051458
© 2005 by the Biophysical Society.

A more recent version of this article appeared on May 1, 2005.
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CELL BIOPHYSICS

High-Resolution Near-Field Optical Imaging of Single Nuclear Pore Complexes under Physiological Conditions

Christiane Höppener 1, Jan-Peter Siebrasse 2, Reiner Peters 2, Ulrich Kubitscheck 3 and Andreas Naber 1*

1 Universität Karlsruhe (TH)
2 Universität Münster
3 Universität Bonn

* To whom correspondence should be addressed. E-mail: andreas.naber{at}physik.uni-karlsruhe.de.

Submitted on August 16, 2004
Revised on October 29, 2004
Accepted on 31 January 2005


  Abstract
Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conven-tional light microscopy and is able to achieve optical resolutions substantially below 100 nm. However, in the field of cell biology SNOM has been rarely applied, probably because previ-ous techniques for sample-distance control are less sensitive in liquid than in air. Recently we developed a distance control based on a tuning fork in tapping mode which is also well suited for imaging in solution. Here we show that this approach can be used to visualize single membrane protein complexes kept in physiological media throughout. Nuclear envelopes (NE) were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated NE were fluorescently la-beled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of ~60 nm. The lateral distribution of epitopes within the supramolecular NPC could be inferred from an analysis of the intensity distribution of the fluorescence spots. The different number densities of p62- and NUP153-labeled NPCs are determined and discussed. Thus we show that SNOM opens up new possibilities for directly visualizing the transport of single particles through single NPCs and other transporters.

Key Words: SNOM in liquid, membrane proteins, nano-optics, nucleocytoplasmic transport, scanning probe microscopy, supramolecular complexes




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Copyright © 2005 by the Biophysical Society.