A 4Pi microscope is a laser scanning
fluorescence microscope
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microsc ...
with an improved
axial resolution
Resolution(s) may refer to:
Common meanings
* Resolution (debate), the statement which is debated in policy debate
* Resolution (law), a written motion adopted by a deliberative body
* New Year's resolution, a commitment that an individual mak ...
. 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
Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser confocal scanning microscopy (LCSM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a sp ...
.
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 In optics, optical path length (OPL, denoted ''Λ'' in equations), also known as optical length or optical distance, is the product of the geometric length of the optical path followed by light and the refractive index of homogeneous medium through ...
through each of the two
objective lens
In optical engineering, the objective is the optical element that gathers light from the object being observed and Focus (optics), focuses the ray (optics), light rays to produce a real image. Objectives can be a single Lens (optics), lens or mirr ...
es 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
In geometry, a solid angle (symbol: ) is a measure of the amount of the field of view from some particular point that a given object covers. That is, it is a measure of how large the object appears to an observer looking from that point.
The poi ...
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.
![Color-online-Simplified-schematic-of-a-4Pi-Type-C-microscope-Excitation-and-detection](https://upload.wikimedia.org/wikipedia/commons/3/32/Color-online-Simplified-schematic-of-a-4Pi-Type-C-microscope-Excitation-and-detection.png)
The operation mode of a 4Pi microscope is shown in the figure. The laser light is divided by a
beam splitter
A beam splitter or ''beamsplitter'' is an optical device that splits a beam of light into a transmitted and a reflected beam. It is a crucial part of many optical experimental and measurement systems, such as interferometers, also finding wide ...
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
. 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
Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness, with 0.64 μm lateral and 3.35 μm axial spatial resolution. Unlike traditional flu ...
is used in a 4Pi microscope in combination with an emission pinhole to lower these side lobes to a tolerable level.
History
In 1971,
Christoph Cremer
Christoph Cremer (born in Freiburg im Breisgau, Germany) is a German physicist and emeritus at the Ruprecht-Karls-University Heidelberg, former honorary professor at the University of Mainz and was a former group leader at Institute of Molecular Bi ...
and
Thomas Cremer
Thomas Cremer (born 7 July 1945 in Miesbach, Germany ), is a German professor of human genetics and anthropology with a main research focus on molecular cytogenetics and 3D/4D analyses of nuclear structure studied by fluorescence microscopy inclu ...
proposed the creation of a perfect
hologram
Holography is a technique that enables a wavefront to be recorded and later re-constructed. Holography is best known as a method of generating real three-dimensional images, but it also has a wide range of other Holography#Applications, applic ...
, i.e. one that carries the whole field information of the emission of a point source in all directions, a so-called
hologram. However the publication from 1978 had drawn an improper physical conclusion (i.e. a point-like spot of light) and had completely missed the axial resolution increase as the actual benefit of adding the other side of the solid angle. The first description of a practicable system of 4Pi microscopy, i.e. the setup with two opposing, interfering lenses, was invented by
Stefan Hell
Stefan Walter Hell HonFRMS (: born 23 December 1962) is a Romanian-German physicist and one of the directors of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany. He received the Nobel Prize in Chemistry in 2014 "for the d ...
in 1991. He demonstrated it experimentally in 1994.
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
mitochondria
A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
in yeast cells with a 4Pi microscope in 2002. A commercial version was launched by microscope manufacturer
Leica Microsystems
Leica Microsystems GmbH is a German microscope manufacturing company. It is a manufacturer of optical microscopes, equipment for the preparation of microscopic specimens and related products. There are ten plants in eight countries with distribu ...
in 2004 and later discontinued.
Up to now, the best quality in a 4Pi microscope was reached in conjunction with super-resolution techniques like the
stimulated emission depletion (STED) principle. Using a 4Pi microscope with appropriate excitation and de-excitation beams, it was possible to create a uniformly 50 nm sized spot, which corresponds to a decreased focal volume compared to confocal microscopy by a factor of 150–200 in fixed cells. With the combination of 4Pi microscopy and
RESOLFT microscopy with switchable proteins, it is now possible to take images of living cells at low light levels with isotropic resolutions below 40 nm.
See also
*
Stimulated emission depletion microscope (STED)
*
Multifocal plane microscopy
Multifocal plane microscopy (MUM) or multiplane microscopy or multifocus microscopy is a form of light microscopy that allows the tracking of the 3D dynamics in live cells at high temporal and spatial resolution by simultaneously imaging differe ...
(MUM)
References
{{DEFAULTSORT:4pi Microscope
Microscopes
Fluorescence
Cell imaging
Laboratory equipment
Microscopy
Optical microscopy techniques