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.