4Pi Microscope - Working Principle

Working Principle

The improvement in resolution is achieved by using two opposing objective lenses which both are focused to the same geometrical location. Also the difference in optical path length through each of the two objective lenses is carefully aligned to be minimal. By this, molecules residing in the common focal area of both objectives can be illuminated coherently from both sides and also the reflected or emitted light can be collected coherently, i.e. coherent superposition of emitted light on the detector is possible. The solid angle that is used for illumination and detection is increased and approaches the ideal case. In this case the sample is illuminated and detected from all sides simultaneously.

The operation mode of a 4Pi microscope is shown in the figure. The laser light is divided by a beam splitter (BS) and directed by mirrors towards the two opposing objective lenses. At the common focal point superposition of both focused light beams occurs. 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 (DM) onto a detector. There superposition of both emitted light pathways can take place again.

In the ideal case each objective lens can collect light from a solid angle of . So, with two objective lenses one can collect from every direction (solid angle ). The name of this type of microscopy is derived from the maximal possible solid angle for excitation and detection. Practically one can achieve only aperture angles of about 140° for an objective lens, which corresponds to about .

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 confocal microscopy).

In a real 4Pi microscope light is not applied from all sides, 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.