4Pi microscopy

 

 

4Pi microscopy provides superresolution along the z-axis by utilizing two opposing objective lenses. This configuration leads to sharpening of the main focal spot by a factor of 5-7 compared to conventional confocal microscopy. Our microscope is a commercial 4Pi microscope (TCS 4Pi microscope of type A, Leica Microsystems) which is only available at a few places worldwide. The system is based on a confocal laser scanning microscope of type TCS SP2 incorporating both one and two photon excitation, photon counting by avalanche photodiodes, equipped with an additional 4Pi attachment. For two photon excitation a modelocked Ti:Sapphire Laser (MaiTai, Spectra Physics) is used.

Analysis can be performed using oil (100x, NA 1.46) or water (63x, NA 1.2) immersion lenses. The axial resolution is 100-120 nm with oil immersion and 150-170 nm with water immersion lenses. Localization accuracy is around 10 nm.

 

A sample of 4Pi measurement:

Imaging of the nuclear pore complex protein Nup358 in whole nuclei of HeLa cells with 4Pi microscopy.

For details see Hüve J, Wesselmann R, Kahms M, Peters R. 4Pi microscopy of the nuclear pore complex. Biophys J 2008;95:877-885