4Pi microscope

A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy. A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy. 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 method, molecules residing in the common focal area of both objectives can be illuminated coherently from both sides and the reflected or emitted light can also be collected coherently, i.e. coherent superposition of emitted light on the detector is possible. The solid angle Ω {displaystyle Omega } that is used for illumination and detection is increased and approaches its maximum. 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 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 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 Ω = 2 π {displaystyle Omega =2pi } . With two objective lenses one can collect from every direction (solid angle Ω = 4 π {displaystyle Omega =4pi } ). 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 Ω ≈ 1.3 π {displaystyle Omega approx 1.3pi } .