SINGLE NUCLEAR-PORES VISUALIZED BY CONFOCAL MICROSCOPY AND IMAGE-PROCESSING

How nuclear pore complexes, mediating the transport of nucleic acids, proteins, and metabolites between cell nucleus and cytoplasm, are arra nged in the nuclear envelope is essentially unknown. Here we describe a method combining high-resolution confocal imaging with image process ing and pattern recognition to visualize single nuclear pore complexes (120 nm diameter), determine their relative positions with nanometer accuracy, and analyze their distribution in situ. The method was teste d by means of a model system in which the very same sample areas could be imaged by confocal and electron microscopy. It was thus found that single fluorescent beads of 105 nm nominal diameter could be localize d with a lateral accuracy of <20 nm and an axial accuracy of similar t o 20 nm. The method was applied to digitonin-permeabilized 3T3 cells, whose nuclear pore complexes were fluorescently labeled with the anti- nucleoporin antibody mAb414. Stacks of optical sections were generated by confocal imaging at high resolution, Herein the nuclear pore compl exes appeared as bright diffraction-limited spots whose centers were l ocalized by fitting them by three-dimensional gaussians, The nearest-n eighbor distribution function and the pair correlation function were c alculated and found to agree well with those of randomly distributed h ard cylinders of 138 +/- 17 nm diameter, but not with those of randoml y distributed points or nonrandomly distributed cylinders. This was su pported by a cluster analysis. Implications for the direct observation of the transport of single particles and molecules through individual nuclear pore complexes are discussed.