4Pi microscope
A 4Pi microscope is a variation of the confocal microscope, which has a higher resolution than the usual normal confocal microscopes resolution of about 200 nm in the lateral and 500-700 nm in the axial direction. The 4Pi microscope can improve the axial resolution to approximately 100-150 nm, the lateral (lateral ) resolution is, however, not changed. Thus, it reaches a nearly spherical focal spot with a total of 5 - 7-fold lower volume.
Principle of operation
The increase in resolution is achieved by the use of two opposite lenses which not only on two sides of coherent light on the preparation, but the light reflected from the specimen, or to collect light emitted by a coherent both sides. The solid angle, which is used for illumination and detection, is increased in this way and approaches the ideal case, Then is illuminated from all directions, and detects light in all directions. The operation of a 4Pi microscope is shown in the figure. The light of a laser is split by a beam splitter ( BS) in two directions and directed by mirrors to two opposite lenses. This focus the light to the same location where the interference occurs. Excited molecules at this location may in turn emit light which is collected by the two lenses together in the above-mentioned beam splitter and is directed via a dichroic mirror (DM) to the detector where the detected light may then interfere.
Could theoretically pro lens light of a half-space, ie, from the solid angle, are collected, so that the two lenses in the entire space () emitted light could be collected. Therefore, the name of this Mikroskopieart is derived from the maximum possible solid angle for excitation and detection. Practically, a detection in all directions can not be achieved. Modern microscope lenses have only a maximum opening angle of 140 °, which corresponds to a solid angle of approx.
3 is different types (A, B, C), depending on whether the two lenses are used for both ( C) on the pickup ( A) for the detection (B) or. The complexity of the microscope thereby increases the Type C, to, in which the coherent Überlagung Objektivfoki the two must be achieved both in the excitation as well as during detection.
The 4Pi microscope has found particular applications in cell biology, since many structures in the order of 200 nm and below. Three-dimensional reconstructions of cells could be significantly improved because the disadvantage of confocal microscopy, the poor resolution along the optical axis, is completely eliminated. In combination with STED microscopy then a nearly spherical focus could be produced with greatly increased resolution.
Development
Published in 1971, Thomas Cremer and Christoph Cremer theoretical calculations on the production of an ideal Holograms to overcome the diffraction limit, which holds an interference field in all directions in space, called a Hologram.
The first description of a practicable method for 4Pi microscopy succeeded Stefan Hell 1991. It includes the two opposing lenses and the use of interference.
In 1994 he succeeded the first practical demonstration of the improved resolution of a 4Pi microscope.
In the following years, the applications of the 4Pi microscope were further improved. With a parallel excitation and detection of molecules in a 4Pi microscope at 64 locations in the specimen at the same time was 2002, the dynamics of the mitochondria are added to yeast cells, since their magnitude is resolvable in the area of a 4Pi microscope.
A commercial version of the 4Pi microscope was brought from Leica Microsystems launched in 2004.
The highlight in the development of optical 4Pi system marked the combination with the STED principle. This made it possible to achieve a uniform small diameter about 50 nm light spot as the focus of a microscope, which roughly corresponds to a volume reduction of focus compared to the standard confocal microscopy by a factor of 150-200 succeeded.