4Pi microscopy of the nuclear pore complex
Jana Hueve 1, Ramona Wesselmann 1, Martin Kahms 1 and Reiner Peters 1*
1 University of Muenster
* To whom correspondence should be addressed. E-mail: petersr{at}uni-muenster.de.
Submitted on December 11, 2007
Revised on March 2, 2008
Accepted on 12 March 2008
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Abstract |
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To explore whether super-resolution fluorescence microscopy is able to resolve topographic features of single cellular protein complexes a two-photon 4Pi microscope was employed to study the nuclear pore complex (NPC). The microscope had an axial resolution of 110-130 nm and a two-color localization accuracy of 5-10 nm. In immune labeled HeLa cells NPCs could be resolved much better by 4Pi than by confocal microscopy. When two epitopes of the NPC, one localized at the tip of the cytoplasmic filaments and the other at the ring of the nuclear basket, were immune labeled, they could be clearly resolved in single NPCs and their distance determined to be 152±30 nm. In cells expressing a Green Fluorescent Protein construct localized at the NPC center the distances between the ring of the nuclear filaments and the NPC center was 76±12 (PtK2 cells) or 91±21 nm (NRK cells) while the distance between the NPC center and the tips of the cytoplasmic filaments was 84±18 nm, all values in good agreement with previous electron or single-molecule fluorescence estimates. We conclude that super-resolution fluorescence microscopy is a powerful method for analyzing single protein complexes and the cellular nanomachinery in general.
Key Words:
4Pi microscopy, nuclear pore complex, optical nanoscopy, single particle localization, topography of protein complexes
