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Laser Scanning 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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Fluorescent And Confocal Microscopes
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum (invisible to the human eye), while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after. Fluorescence has many practical applications, including mineralogy, gemology, medicine, chemical sensors (fluorescence spectroscopy), fluorescent labelling, dyes, biological detectors, cosmic-ray detection, vacuu ...
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Green Fluorescent Protein
The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aequorea victoria'' and is sometimes called ''avGFP''. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets. The GFP from ''A. victoria'' has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from the sea pansy ('' Renilla reniformis'') has a single major excitation peak at 498 nm. GFP makes for an excellent tool in many forms of biology due to its ability to form an internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than mol ...
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Transgenic
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. ''Transgene'' describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that parti ...
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Fluorophore
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds. Fluorophores are sometimes used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator (when its fluorescence is affected by environmental aspects such as polarity or ions). More generally they are covalently bonded to a macromolecule, serving as a marker (or dye, or tag, or reporter) for affine or bioactive reagents ( antibodies, peptides, nucleic acids). Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, i.e., fluorescent imaging and spectroscopy. Fluorescein, via its amine-reactive isothiocyanate derivative fluorescein isothiocyanate (FITC), has been one of the most popular fluorophores. F ...
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Objective Lens
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 propert ...
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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 in ...
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Contrast (vision)
Contrast is the contradiction in luminance or colour that makes an object (or its representation in an image or display) distinguishable. In visual perception of the real world, contrast is determined by the difference in the color, colour and brightness of the object and other objects within the same field of view. The human visual system is more sensitive to contrast than absolute luminance; we can perceive the world similarly regardless of the huge changes in illumination over the day or from place to place. The maximum ''contrast'' of an image is the contrast ratio or dynamic range. Images with a contrast ratio close to their medium's maximum possible contrast ratio experience a ''conservation of contrast'', wherein any increase in contrast in some parts of the image must necessarily result in a decrease in contrast elsewhere. Brightening an image will increase contrast in dark areas but decrease contrast in bright areas, while darkening the image will have the opposite effec ...
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Signal-to-noise Ratio
Signal-to-noise ratio (SNR or S/N) is a measure used in science and engineering that compares the level of a desired signal to the level of background noise. SNR is defined as the ratio of signal power to the noise power, often expressed in decibels. A ratio higher than 1:1 (greater than 0 dB) indicates more signal than noise. SNR, bandwidth, and channel capacity of a communication channel are connected by the Shannon–Hartley theorem. Definition Signal-to-noise ratio is defined as the ratio of the power of a signal (meaningful input) to the power of background noise (meaningless or unwanted input): : \mathrm = \frac, where is average power. Both signal and noise power must be measured at the same or equivalent points in a system, and within the same system bandwidth. Depending on whether the signal is a constant () or a random variable (), the signal-to-noise ratio for random noise becomes: : \mathrm = \frac where E refers to the expected value, i.e. in this ...
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Latency (engineering)
Latency, from a general point of view, is a time delay between the cause and the effect of some physical change in the system being observed. Lag, as it is known in gaming circles, refers to the latency between the input to a simulation and the visual or auditory response, often occurring because of network delay in online games. Latency is physically a consequence of the limited velocity at which any physical interaction can propagate. The magnitude of this velocity is always less than or equal to the speed of light. Therefore, every physical system with any physical separation (distance) between cause and effect will experience some sort of latency, regardless of the nature of the stimulation at which it has been exposed to. The precise definition of latency depends on the system being observed or the nature of the simulation. In communications, the lower limit of latency is determined by the medium being used to transfer information. In reliable two-way communication ...
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Servomechanism
In control engineering a servomechanism, usually shortened to servo, is an automatic device that uses error-sensing negative feedback to correct the action of a mechanism. On displacement-controlled applications, it usually includes a built-in encoder or other position feedback mechanism to ensure the output is achieving the desired effect. The term correctly applies only to systems where the feedback or error-correction signals help control mechanical position, speed, attitude or any other measurable variables. For example, an automotive power window control is not a servomechanism, as there is no automatic feedback that controls position—the operator does this by observation. By contrast a car's cruise control uses closed-loop feedback, which classifies it as a servomechanism. Applications Position control A common type of servo provides ''position control''. Commonly, servos are electric, hydraulic, or pneumatic. They operate on the principle of negative feedback ...
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Avalanche Photodiode
An avalanche photodiode (APD) is a highly sensitive semiconductor photodiode detector that exploits the photoelectric effect to convert light into electricity. From a functional standpoint, they can be regarded as the semiconductor analog of photomultiplier tubes. The avalanche photodiode (APD) was invented by Japanese engineer Jun-ichi Nishizawa in 1952. However, study of avalanche breakdown, microplasma defects in silicon and germanium and the investigation of optical detection using p-n junctions predate this patent. Typical applications for APDs are laser rangefinders, long-range fiber-optic telecommunication, and quantum sensing for control algorithms. New applications include positron emission tomography and particle physics. It was discovered in 2020 that adding graphene layer can prevent degradation over time to keep avalanche photodiode''like new'' which is important in shrinking their size and costs for many diverse applications & bringing devices out of vacuum tubes in ...
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