4Pi Microscope

A 4Pi microscope is a confocal microscope with two opposing lenses. It is used for high resolution imaging of fluorescence.

It can be operated in three different ways: In a 4Pi microscope of type A, the coherent superposition of excitation light is used to generate the increased resolution. The emission light is either detected from one side only or in an incoherent superposition from both sides. In a 4Pi microscope of Type B, only the emission light is interfering. When operated in the Type C mode, both excitation and emission light are allowed to interfere leading to the highest possible resolution increase (~7 fold along the optic axis as compared to wide field fluorescence microscopy). A typical axial resolution of a 4Pi microscope is about 100 nm.

Typically (but not inevitably) two-photon excitation microscopy is used in a 4Pi microscope in combination with the emission pinhole to lower the side lobes of the point spread function.

The method was pioneered by Stefan Hell and his co-workers. It is related to the method of I5 microscopy pioneered by Mats Gustafsson.

4Pi-Mikroskop