4Pi microscope

Excited molecules at this position emit fluorescence light, which is collected by both objective lenses, combined by the same beam splitter and deflected by a dichroic mirror onto a detector.

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 optical axis as compared to confocal microscopy).

In a real 4Pi microscope light cannot be applied or collected from all directions equally, leading to so-called side lobes in the point spread function.

Typically (but not always) two-photon excitation microscopy is used in a 4Pi microscope in combination with an emission pinhole to lower these side lobes to a tolerable level.

[2][3] However the publication from 1978 [4] had drawn an improper physical conclusion (i.e. a point-like spot of light) and had completely missed the axial resolution increase as the actual benefit of adding the other side of the solid angle.

[5] The first description of a practicable system of 4Pi microscopy, i.e. the setup with two opposing, interfering lenses, was invented by Stefan Hell in 1991.

For example, parallel excitation and detection with 64 spots in the sample simultaneously combined with the improved spatial resolution resulted in the successful recording of the dynamics of mitochondria in yeast cells with a 4Pi microscope in 2002.

Up to now, the best quality in a 4Pi microscope was reached in conjunction with super-resolution techniques like the stimulated emission depletion (STED) principle.

[10] Using a 4Pi microscope with appropriate excitation and de-excitation beams, it was possible to create a uniformly 50 nm sized spot, which corresponds to a decreased focal volume compared to confocal microscopy by a factor of 150–200 in fixed cells.