Confocal microscopy is an optical imaging technique used to increase
micrographA micrograph, or photomicrograph is a photograph or similar image taken through a microscope or similar device to show a magnified image of an item. Canadian inventor Reginald Aubrey Fessenden is credited with inventing photomicrography....
contrastContrast is the difference in visual properties that makes an object distinguishable from other objects and the background. In visual perception of the real world, contrast is determined by the difference in the color and brightness of the object and other objects within the same field of view...
and/or to reconstruct three-dimensional
imageAn image is an artifact, for example a two-dimensional picture, that has a similar appearance to some subject—usually a physical object or a person....
s by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. This technique has gained popularity in the scientific and industrial communities and typical applications are in
life sciencesWe define Life Sciences to encompass companies in the fields of biotechnology, pharmaceuticals, biomedical technologies, life systems technologies, nutraceuticals, cosmeceuticals, food processing, environmental, biomedical devices, and organizations and institutions that devote the majority of...
and
semiconductorA semiconductor is a material that has an electrical resistivity between that of a conductor and an insulator, that is, generally in the range 10
3 Siemens/cm to 10
−8 S/cm. Devices made from semiconductor materials are the foundation of modern electronics, including radio,...
inspection.
The principle of confocal imaging was patented by
Marvin MinskyMarvin Lee Minsky is an American cognitive scientist in the field of artificial intelligence , co-founder of Massachusetts Institute of Technology's AI laboratory, and author of several texts on AI and philosophy.-Biography:...
in 1957 and aims to overcome some limitations of traditional wide-field
fluorescence microscopeA fluorescence microscope is a light 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.- Technique :In most cases, a component of interest in the specimen is...
s.
Confocal microscopy is an optical imaging technique used to increase
micrographA micrograph, or photomicrograph is a photograph or similar image taken through a microscope or similar device to show a magnified image of an item. Canadian inventor Reginald Aubrey Fessenden is credited with inventing photomicrography....
contrastContrast is the difference in visual properties that makes an object distinguishable from other objects and the background. In visual perception of the real world, contrast is determined by the difference in the color and brightness of the object and other objects within the same field of view...
and/or to reconstruct three-dimensional
imageAn image is an artifact, for example a two-dimensional picture, that has a similar appearance to some subject—usually a physical object or a person....
s by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. This technique has gained popularity in the scientific and industrial communities and typical applications are in
life sciencesWe define Life Sciences to encompass companies in the fields of biotechnology, pharmaceuticals, biomedical technologies, life systems technologies, nutraceuticals, cosmeceuticals, food processing, environmental, biomedical devices, and organizations and institutions that devote the majority of...
and
semiconductorA semiconductor is a material that has an electrical resistivity between that of a conductor and an insulator, that is, generally in the range 10
3 Siemens/cm to 10
−8 S/cm. Devices made from semiconductor materials are the foundation of modern electronics, including radio,...
inspection.
Basic concept
The principle of confocal imaging was patented by
Marvin MinskyMarvin Lee Minsky is an American cognitive scientist in the field of artificial intelligence , co-founder of Massachusetts Institute of Technology's AI laboratory, and author of several texts on AI and philosophy.-Biography:...
in 1957 and aims to overcome some limitations of traditional wide-field
fluorescence microscopeA fluorescence microscope is a light 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.- Technique :In most cases, a component of interest in the specimen is...
s. In a conventional (i.e., wide-field)
fluorescence microscopeA fluorescence microscope is a light 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.- Technique :In most cases, a component of interest in the specimen is...
, the entire
specimenIn biology, a specimen is an individual animal, part of an animal, plant, part of a plant, or microorganism used as a representative to study the properties of the whole population of that species or subspecies...
is flooded in light from a light source. All parts of the specimen in the optical path are excited and the resulting
fluorescenceFluorescence is the emission of visible light by a substance that has absorbed light of a differing, usually invisible, wavelength. Absorption of a photon triggers the emission of a photon with a longer wavelength. A shorter wavelength emission is sometimes observed from multiple photon absorption...
is detected by the microscope
photodetectorPhotosensors or photodetectors are sensors of light or other electromagnetic energy. There are several varieties: *Optical detectors, which are mostly quantum devices in which an individual photon produces a discrete effect....
or
camerathumb |right|Cameras from Large to Small, Film to Digital A camera is a device that records images, either as a still photograph or as moving images known as videos or movies...
as background signal. In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information - the name "confocal" stems from this configuration. As only light produced by fluorescence very close to the focal plane can be detected the image
resolutionResolution may refer to:* Resolution , a rule of inference used for automated theorem proving* Resolution , a written motion adopted by a deliberative body* Resolution , a technique in music theory...
, particularly in the sample depth direction, is much better than that of wide-field microscopes. However as much of the light from sample fluorescence is blocked at the pinhole this increased resolution is at the cost of decreased signal intensity so long
exposureIn photography, exposure is the total amount of light allowed to fall on the photographic medium during the process of taking a photograph...
s are often required.
As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The thickness of the focal plane is defined mostly by the
inverseIn mathematics, two quantities are said to be proportional if they vary in such a way that one of the quantities is a constant multiple of the other, or equivalently if they have a constant ratio.Proportion also refers to the equality of two ratios....
of the square of the
numerical apertureIn optics, the numerical aperture of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light...
of the objective lens, and also by the optical properties of the specimen and the ambient index of refraction. The thin
optical sectioningOptical sectioning is the process by which a suitably designed microscope can produce clear images of a focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome...
possible make these types of microscopes particularly good at 3D imaging of samples.
Types
Three types of confocal microscopes are commercially available:
- Confocal laser scanning microscopes
- Spinning-disk (Nipkow disk
A Nipkow disk , also known as scanning disk, is a mechanical, geometrically operating image scanning device, invented by Paul Gottlieb Nipkow...
) confocal microscopes
- Programmable Array Microscopes (PAM)
Each of these classes of confocal microscope have particular advantages and disadvantages, most systems are either optimised for resolution or high sensitivity for video capture. Confocal laser scanning microscopes generally yield better image quality than Nipkow and PAM but imaging
frame rateFrame rate, or frame frequency, is the measurement of the frequency at which an imaging device produces unique consecutive images called frames. The term applies equally well to computer graphics, video cameras, film cameras, and motion capture systems...
s are typically very slow (less than 3 frames/second). Spinning-disk confocal microscopes can achieve video rate imaging - a desirable feature for dynamic observations such as live cell imaging - but at lower resolution.
Cutting edge development of confocal laser scanning microscopy now allows better than video rate (60 frames/second) imaging by using multiple MEMS based scanning mirrors.
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