High-resolution near-field optical imaging of single nuclear pore complexes under physiological conditions.
Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conventional 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 previous 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 were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated nuclear envelopes were fluorescently labeled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of approximately 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.
Höppener
C
C
Institut für Angewandte Physik, Universität Karlsruhe, Karlsruhe, Germany.
Siebrasse
J P
JP
Peters
R
R
Kubitscheck
U
U
Naber
A
A
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008856:Microscopy, Fluorescence / Q000379:methods*; D016011:Normal Distribution; D009685:Nuclear Envelope / Q000648:ultrastructure*; D022022:Nuclear Pore / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000096:biosynthesis / Q000737:chemistry; D009865:Oocytes / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*
D000939:Epitopes; D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; C049312:nuclear pore protein p62; D049452:Green Fluorescent Proteins