4Pi Microscope
Encyclopedia
A 4Pi Microscope is a laser scanning fluorescence microscope
with an improved axial
resolution
. The typical value 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
.
through each of the two objective lenses is carefully aligned to be minimal. By this, molecules residing in the common focal area of both objectives can be illuminated coherently from both sides and also the reflected or emitted light can be collected coherently, i.e. coherent superposition of emitted light on the detector is possible. The solid angle
that is used for illumination and detection is increased and approaches the ideal case. 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 (BS) 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 (DM) 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
. So, 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 
about 
.
It 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 optic axis as compared to confocal microscopy).
In a real 4Pi microscope light is not applied from all sides, 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.
and Thomas Cremer
proposed the creation of a perfect hologram, i.e. that carries the whole field information of the emission of a point source in all directions, a so-called
hologram.
The first description of a practicable system of 4Pi microscopy, i.e. the setup with two opposing, interfering lenses, was invented by Stefan Hell
in 1991. He could demonstrate it experimentally also in 1994.
In the following years the number of applications for this microscope have grown. 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 in yeast cells with a 4Pi microscope in 2002.
A commercial version was launched by microscope manufacturer Leica Microsystems
in 2004.
Up to now, the best quality in a 4Pi microscope was reached in conjunction with the 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.
Fluorescence microscope
A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption...
with an improved axial
Optical axis
An optical axis is a line along which there is some degree of rotational symmetry in an optical system such as a camera lens or microscope.The optical axis is an imaginary line that defines the path along which light propagates through the system...
resolution
Optical resolution
Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged.An imaging system may have many individual components including a lens and recording and display components...
. The typical value 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
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...
.
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 lengthOptical path length
In optics, optical path length or optical distance is the product of the geometric length of the path light follows through the system, and the index of refraction of the medium through which it propagates. A difference in optical path length between two paths is often called the optical path...
through each of the two objective lenses is carefully aligned to be minimal. By this, molecules residing in the common focal area of both objectives can be illuminated coherently from both sides and also the reflected or emitted light can be collected coherently, i.e. coherent superposition of emitted light on the detector is possible. The solid angle
Solid angle
The solid angle, Ω, is the two-dimensional angle in three-dimensional space that an object subtends at a point. It is a measure of how large that object appears to an observer looking from that point...
that is used for illumination and detection is increased and approaches the ideal case. 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 (BS) 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 (DM) 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
. So, 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 about .It 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 optic axis as compared to confocal microscopy).
In a real 4Pi microscope light is not applied from all sides, leading to so-called side lobes in the point spread function
Point spread function
The point spread function describes the response of an imaging system to a point source or point object. A more general term for the PSF is a system's impulse response, the PSF being the impulse response of a focused optical system. The PSF in many contexts can be thought of as the extended blob...
. Typically (but not always) two-photon excitation microscopy
Two-photon excitation microscopy
Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite...
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 CremerChristoph Cremer
Christoph Cremer is a German physicist and professor at the Ruprecht-Karls-University Heidelberg, who has successfully overcome the conventional limit of resolution that applies to light based investigations by a range of different methods Christoph Cremer (born in Freiburg im Breisgau, Germany)...
and Thomas Cremer
Thomas Cremer
Thomas Cremer , 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 confocal microscopy and live cell imaging...
proposed the creation of a perfect hologram, i.e. that carries the whole field information of the emission of a point source in all directions, a so-called
hologram.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 Hell
Stefan W. Hell is a physicist and one of the directors of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany as well as the head of the department "Optical Nanoscopy" at the German Cancer Research Center in Heidelberg.- Life :In 1981 Hell began his studies at the...
in 1991. He could demonstrate it experimentally also in 1994.
In the following years the number of applications for this microscope have grown. 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 in yeast cells with a 4Pi microscope in 2002.
A commercial version was launched by microscope manufacturer Leica Microsystems
Leica Microsystems
Leica Microsystems GmbH is a leading global manufacturer of optical microscopes, equipment for the preparation of microscopic specimens and related products. There are ten plants in eight countries with distribution partners in over 100 countries...
in 2004.
Up to now, the best quality in a 4Pi microscope was reached in conjunction with the 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.