4pi microscopy of the nuclear pore complex

Kahms M., Hüve J., Peters R.

Abstract

4Pi microscopy is a far-fi eld fl uorescence microscopy technique, in which the wave fronts of two opposing illuminating beams are adjusted to constructively interfere in a common focus. This yields a diffraction pattern in the direction of the optical axis, which essentially consists of a main focal spot accompanied by two smaller side lobes. At optimal conditions, the main peak of this so-called point spread function has a full width at half maximum: fi xed phrase of 100 nm in the direction of the optical axis, and thus is 6–7-fold smaller than that of a confocal microscope. In this chapter, we describe the basic features of 4Pi microscopy and its application to cell biology using the example of the nuclear pore complex, a large protein assembly spanning the nuclear envelope.