Fluorescent Microscopy
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Fluorescent Microscopy
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 microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image. Principle The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter. Typical components of a fluorescence microscope are a light source (xenon arc lamp or mercury-vapor lamp are ...
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Excitation Filter
An excitation filter is a high quality optical-glass filter commonly used in fluorescence microscopy and spectroscopic applications for selection of the excitation wavelength of light from a light source. Most excitation filters select light of relatively short wavelengths from an excitation light source, as only those wavelengths would carry enough energy to cause the object the microscope is examining to fluoresce sufficiently. The excitation filters used may come in two main types — short pass filters and band pass filters. Variations of these filters exist in the form of notch filters or deep blocking filters (commonly employed as emission filters Emission may refer to: Chemical products * Emission of air pollutants, notably: **Flue gas, gas exiting to the atmosphere via a flue ** Exhaust gas, flue gas generated by fuel combustion ** Emission of greenhouse gases, which absorb and emit rad ...). Other forms of excitation filters include the use of monochromators, wedge prisms ...
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Dichroic
In optics, a dichroic material is either one which causes visible light to be split up into distinct beams of different wavelengths (colours) (not to be confused with dispersion), or one in which light rays having different polarizations are absorbed by different amounts. In beam splitters The original meaning of ''dichroic'', from the Greek ''dikhroos'', two-coloured, refers to any optical device which can split a beam of light into two beams with differing wavelengths. Such devices include mirrors and filters, usually treated with optical coatings, which are designed to reflect light over a certain range of wavelengths and transmit light which is outside that range. An example is the dichroic prism, used in some camcorders, which uses several coatings to split light into red, green and blue components for recording on separate CCD arrays, however it is now more common to have a Bayer filter to filter individual pixels on a single CCD array. This kind of dichroic device does n ...
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Total Internal Reflection Fluorescence Microscope
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid-liquid interface with the same frequency as the excitation light. The intensity of the evanescent wave exponentially decays with distance from the surface of the solid so that only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. Two-dimensional images of the fluorescence can then be obtained, although there are also mechanisms in which three-dimensional information on th ...
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Supercontinuum
In optics, a supercontinuum is formed when a collection of nonlinear processes act together upon a pump beam in order to cause severe spectral broadening of the original pump beam, for example using a microstructured optical fiber. The result is a smooth spectral continuum (see figure 1 for a typical example). There is no consensus on how much broadening constitutes a supercontinuum; however researchers have published work claiming as little as 60 nm of broadening as a supercontinuum. There is also no agreement on the spectral flatness required to define the bandwidth of the source, with authors using anything from 5 dB to 40 dB or more. In addition the term supercontinuum itself did not gain widespread acceptance until this century, with many authors using alternative phrases to describe their continua during the 1970s, 1980s and 1990s. During the last decade, the development of supercontinua sources has emerged as a research field. This is largely due to ne ...
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Halogen Lamp
A halogen lamp (also called tungsten halogen, quartz-halogen, and quartz iodine lamp) is an incandescent lamp consisting of a tungsten filament sealed in a compact transparent envelope that is filled with a mixture of an inert gas and a small amount of a halogen, such as iodine or bromine. The combination of the halogen gas and the tungsten filament produces a halogen-cycle chemical reaction, which redeposits evaporated tungsten on the filament, increasing its life and maintaining the clarity of the envelope. This allows the filament to operate at a higher temperature than a standard incandescent lamp of similar power and operating life; this also produces light with higher luminous efficacy and color temperature. The small size of halogen lamps permits their use in compact optical systems for projectors and illumination. The small glass envelope may be enclosed in a much larger outer glass bulb, which has a lower temperature, protects the inner bulb from contamination, and mak ...
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Numerical Aperture
In optics, the numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the property that it is constant for a beam as it goes from one material to another, provided there is no refractive power at the interface. The exact definition of the term varies slightly between different areas of optics. Numerical aperture is commonly used in microscopy to describe the acceptance cone of an objective (and hence its light-gathering ability and resolution), and in fiber optics, in which it describes the range of angles within which light that is incident on the fiber will be transmitted along it. General optics In most areas of optics, and especially in microscopy, the numerical aperture of an optical system such as an objective lens is defined by :\mathrm = n \sin \theta, where is the index of refraction of the medium i ...
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Objective (optics)
In optical engineering, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be a single lens or mirror, or combinations of several optical elements. They are used in microscopes, binoculars, telescopes, cameras, slide projectors, CD players and many other optical instruments. Objectives are also called object lenses, object glasses, or objective glasses. Microscope objectives The objective lens of a microscope is the one at the bottom near the sample. At its simplest, it is a very high-powered magnifying glass, with very short focal length. This is brought very close to the specimen being examined so that the light from the specimen comes to a focus inside the microscope tube. The objective itself is usually a cylinder containing one or more lenses that are typically made of glass; its function is to collect light from the sample. Magnification One of the most important prope ...
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Life Sciences
This list of life sciences comprises the branches of science that involve the scientific study of life – such as microorganisms, plants, and animals including human beings. This science is one of the two major branches of natural science, the other being physical science, which is concerned with non-living matter. Biology is the overall natural science that studies life, with the other life sciences as its sub-disciplines. Some life sciences focus on a specific type of organism. For example, zoology is the study of animals, while botany is the study of plants. Other life sciences focus on aspects common to all or many life forms, such as anatomy and genetics. Some focus on the micro-scale (e.g. molecular biology, biochemistry) other on larger scales (e.g. cytology, immunology, ethology, pharmacy, ecology). Another major branch of life sciences involves understanding the mindneuroscience. Life sciences discoveries are helpful in improving the quality and standard of life and h ...
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Total Internal Reflection Fluorescence Microscope
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid-liquid interface with the same frequency as the excitation light. The intensity of the evanescent wave exponentially decays with distance from the surface of the solid so that only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. Two-dimensional images of the fluorescence can then be obtained, although there are also mechanisms in which three-dimensional information on th ...
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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 spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science. Light travels through the sample under a conventional microscope as far into the specimen as it can penetrate, while a confocal microscope only focuses a smaller beam of light at one narrow depth level at a time. The CLSM achieves a controlled and highly limited depth of field. Basic concept The principle of co ...
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