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.

Scanning near-field optical microscopy of a cell membrane in liquid.

The applications of scanning near-field optical microscopy to biological specimens under physiological conditions have so far been very rare since common techniques for a probe-sample distance control are not as well suited for operation in liquid as under ambient conditions. We have shown previously that our own approach for a distance control, based on a short aperture fibre probe and a tuning fork as force sensor in a tapping mode, works well even on soft material in water. By means of an electronic self-excitation circuit, which compensates for changes of the resonance frequency due to evaporation of liquid, the stability of the force feedback has now been further improved. We present further evidence for the excellent suitability of the tapping-mode-like distance control to an operation in liquid, for example, by force-imaging of double-stranded DNA. Moreover, we demonstrate that a nuclear envelope in liquid can be imaged with a high optical resolution of approximately 70 nm without affecting its structural integrity. Thereby, single nuclear pores in the nuclear envelope with a nearest neighbour distance of approximately 120 nm have been optically resolved for the first time.

Enhanced light confinement in a near-field optical probe with a triangular aperture.

We present a probe concept for scanning near-field optical microscopy combining the excellent background suppression of aperture probes with the superior light confinement of apertureless probes. A triangular aperture at the tip of a tetrahedral waveguide (full taper angle approximately 90 degrees ) shows a strong field enhancement at only one rim when illuminated with light of suitable polarization. Compared to a circular aperture of equivalent size, the resolution capability is doubled without loss of brightness. For a approximately 60 nm sized triangular aperture, we measured an optical resolution <40 nm and a transmission of approximately 10(-4).