4Pi microscopy of the nuclear pore complex.
4Pi microscopy is a far-field fluorescence 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: fixed 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.
Institute for Medical Physics and Biophysics, University Muenster, Robert-Koch-Strasse 31, Muenster, 48149, USA.
Martin
M
Kahms
Jana
J
Hüve
Reiner
R
Peters
D003584:Cytological Techniques / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D008857:Microscopy, Interference / Q000379:methods*; D022022:Nuclear Pore / Q000648:ultrastructure*