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.[1]
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
The operation mode of a 4Pi microscope is shown in the figure. The laser light is divided by a
In the ideal case each objective lens can collect light from a solid angle of . 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 .
The microscope 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 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.
History
In 1971,
In the following years, the number of applications for this microscope has grown. 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
Up to now, the best quality in a 4Pi microscope was reached in conjunction with super-resolution techniques like the
See also
- Stimulated emission depletion microscope(STED)
- Multifocal plane microscopy (MUM)
References
- ^ J. Bewersdorf; A. Egner; S.W. Hell (2004). "4Pi-Confocal Microscopy is Coming of Age" (PDF). GIT Imaging & Microscopy (4): 24–25.
- ^ Cremer C., Cremer T. (1971) Punkthologramme: Physikalische Grundlagen und mögliche Anwendungen. Enclosure to Patent application DE 2116521 „Verfahren zur Darstellung bzw. Modifikation von Objekt-Details, deren Abmessungen außerhalb der sichtbaren Wellenlängen liegen" (Procedure for the imaging and modification of object details with dimensions beyond the visible wavelengths). Filed April 5, 1971; publication date October 12, 1972. Deutsches Patentamt, Berlin. http://depatisnet.dpma.de/DepatisNet/depatisnet?action=pdf&docid=DE000002116521A
- ^ Considerations on a laser-scanning-microscope with high resolution and depth of field: C. Cremer and T. Cremer in MICROSCOPICA ACTA VOL. 81 NUMBER 1 September, p. 31–44 (1978). Basic design of a confocal laser scanning fluorescence microscope & principle of a confocal laser scanning 4Pi fluorescence microscope, 1978 Archived 2016-03-04 at the Wayback Machine.
- ^ C. Cremer and T. Cremer (1978): Considerations on a laser-scanning-microscope with high resolution and depth of field Microscopica Acta VOL. 81 NUMBER 1 September, pp. 31—44 (1978)
- ^ The Nobel Prize in Chemistry 2014 https://www.nobelprize.org/prizes/chemistry/2014/hell/biographical/
- ^ European patent EP 0491289.
- PMID 19829598.
- PMID 11904401.
- ^ Review article 4Pi microscopy.
- S2CID 16580036.
- PMID 26833381.