4Pi microscope

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A 4Pi microscope is a variant of the confocal microscope , which has a higher resolution than the resolution of about 200 nm in the lateral and 500–700 nm in the axial direction , which is usual with normal confocal microscopes . The 4Pi microscope can improve the axial resolution to around 100–150 nm, but the lateral resolution is not changed. As a result, it achieves an almost spherical focal light spot with a total of 5-7 times less volume.

Working principle

The increase of resolution is through the use of two opposite lenses achieved that the preparation not only from two sides coherent light, but to collect the reflected or emitted by the specimen light from both sides coherent. The solid angle that is used for illumination and detection increases in this way and approaches the ideal case: Then it is illuminated from all spatial directions and light is detected in all spatial directions. How a 4Pi microscope works is shown in the figure. The light from a laser is split into two directions by a beam splitter (BS) and directed to two opposing lenses via mirrors. These focus the light on the same place where the interference occurs. Excited molecules at this location can in turn emit light, which is captured by both lenses, brought together in the aforementioned beam splitter and directed via a dichroic mirror (DM) to the detector, where the detected light can then interfere.

In theory, light from a half-space, i.e. from the solid angle of , could be collected for each lens so that the light emitted into the entire room ( ) could be collected with two lenses . The name of this type of microscopy is derived from the maximum possible solid angle for excitation and detection. In practice, detection cannot be achieved in all directions. Modern microscope objectives only have a maximum opening angle of approx. 140 °, which corresponds to a solid angle of approx .

There are 3 types (A, B, C), depending on whether two objectives are only used for excitation (A), only for detection (B), or for both (C). The complexity of the microscope increases towards type C, in which the coherent superposition of the two objective foci has to be achieved both in the excitation and in the detection.

The 4Pi microscope has found special applications in cell biology, as many structures are 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 , it was then possible to generate an almost spherical focus with a greatly increased resolution.

development

In 1971, Thomas Cremer and Christoph Cremer published theoretical calculations on the creation of an ideal hologram to overcome the diffraction limit, which holds an interference field in all spatial directions, a so-called 4π hologram.

The first description of a practicable method for 4Pi microscopy was made by Stefan Hell in 1991. It includes the two opposing objectives and the use of interference.

In 1994 he also succeeded in the first practical demonstration of the improved resolution of a 4Pi microscope.

In the following years the application possibilities of the 4Pi microscope were further improved. With a parallel excitation and detection of molecules in a 4Pi microscope at 64 points in the preparation, the dynamics of the mitochondria in yeast cells could be recorded in 2002, since their size is in the resolvable range of a 4Pi microscope.

A commercial version of the 4Pi microscope was launched by Leica Microsystems in 2004.

Through the combination of 4Pi optical systems and STED , it was possible in 2009 to achieve a uniform light spot with a diameter of around 50 nm as the focus of a microscope in fixed cells, which is roughly a volume reduction of the focus compared to standard confocal microscopy by a factor of 150– 200 corresponds. The combination of 4Pi microscopy with RESOLFT microscopy with switchable proteins has even made it possible to create images with isotropic resolution in living cells at significantly lower intensities since 2015.

See also

Web links

Individual evidence

  1. ↑ C. Decker: 4Pi Microscopy - Principle and Realization ( Memento from December 22, 2015 in the Internet Archive ). Handout for the seminar presentation on December 14, 2010.
  2. ^ German Patent Office: Offenlegungsschrift from October 12, 1972
  3. ↑ Construction plan 1978: Confocal laser scanning fluorescence microscope with high resolution and depth of field / 4Pi Point Hologram (PDF; 83 kB)
  4. ↑ Patent EP0491289 : Double-confocal scanning microscope. Published on June 24, 1992 , inventor: Stefan Hell. ‌
  5. ↑ SW Hell, EHK Stelzer: Properties of a 4Pi confocal fluorescence microscope . In: Journal of the Optical Society of America A: Optics, Image Science, and Vision . Vol. 9, No. 12 , 1992, pp. 2159-2166 , doi : 10.1364 / JOSAA.9.002159 . SW Hell, EHK Stelzer, S. Lindek, C. Cremer: Confocal microscopy with an increased detection aperture: Type-B 4Pi confocal microscopy . In: Optics Letters . tape
     19 , no. 3 , 1994, p. 222-224 , doi : 10.1364 / OL.19.000222 .
  6. ^ A. Egner, S. Jakobs, SW Hell: Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast . In: PNAS . tape 99 , 2002, pp. 3370-3375 , doi : 10.1073 / pnas.052545099 .
  7. ↑ Leica press release ( Memento from March 1, 2012 in the Internet Archive ) (Eng.) Last saved on March 1, 2012.
  8. ^ R. Schmidt, CA Wurm, S. Jakobs, J. Engelhardt, A. Egner, SW Hell: Spherical nanosized focal spot unravels the interior of cells . In: Nature Methods . tape 5 , 2008, p. 539-544 , doi : 10.1038 / nmeth.1214 .
  9. ↑ Ulrike Böhm, Stefan W. Hell, Roman Schmidt: 4Pi-RESOLFT nanoscopy . In: Nature Communications . Vol. 7, No. 10504 , 2016, p. 1-8 , doi : 10.1038 / ncomms10504 .