Fluorescence microscopeA 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.
Fluorescence spectroscopyFluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.
ImmunofluorescenceImmunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. The specific region an antibody recognizes on an antigen is called an epitope.
FluorophoreA 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).
Green fluorescent proteinThe 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.
PhotobleachingIn optics, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between the fluorophore and surrounding molecules. Such irreversible modifications in covalent bonds are caused by transition from a singlet state to the triplet state of the fluorophores. The number of excitation cycles to achieve full bleaching varies.
Super-resolution microscopySuper-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field (photon-tunneling microscopy as well as those that use the Pendry Superlens and near field scanning optical microscopy) or on the far-field.
FluorescenceFluorescence 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.
MicroscopyMicroscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy. Optical microscopy and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/electron beams interacting with the specimen, and the collection of the scattered radiation or another signal in order to create an image.
Fluorescent tagIn molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically.
Fluorescence in the life sciencesFluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence (such as NADH, tryptophan or endogenous chlorophyll, phycoerythrin or green fluorescent protein). Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot.
Cross-linkIn chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers (such as proteins). In polymer chemistry "cross-linking" usually refers to the use of cross-links to promote a change in the polymers' physical properties. When "crosslinking" is used in the biological field, it refers to the use of a probe to link proteins together to check for protein–protein interactions, as well as other creative cross-linking methodologies.
Multispectral imagingMultispectral imaging captures image data within specific wavelength ranges across the electromagnetic spectrum. The wavelengths may be separated by filters or detected with the use of instruments that are sensitive to particular wavelengths, including light from frequencies beyond the visible light range, i.e. infrared and ultra-violet. It can allow extraction of additional information the human eye fails to capture with its visible receptors for red, green and blue.
Förster resonance energy transferFörster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.
Confocal microscopyConfocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), 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.
Flow cytometryFlow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths.
Electron-beam processingElectron-beam processing or electron irradiation (EBI) is a process that involves using electrons, usually of high energy, to treat an object for a variety of purposes. This may take place under elevated temperatures and nitrogen atmosphere. Possible uses for electron irradiation include sterilization, alteration of gemstone colors, and cross-linking of polymers. Electron energies typically vary from the keV to MeV range, depending on the depth of penetration required.
Hyperspectral imagingHyperspectral imaging collects and processes information from across the electromagnetic spectrum. The goal of hyperspectral imaging is to obtain the spectrum for each pixel in the image of a scene, with the purpose of finding objects, identifying materials, or detecting processes. There are three general types of spectral imagers. There are push broom scanners and the related whisk broom scanners (spatial scanning), which read images over time, band sequential scanners (spectral scanning), which acquire images of an area at different wavelengths, and snapshot hyperspectral imagers, which uses a staring array to generate an image in an instant.
Cross-linked polyethyleneCross-linked polyethylene, commonly abbreviated PEX, XPE or XLPE, is a form of polyethylene with cross-links. It is used predominantly in building services pipework systems, hydronic radiant heating and cooling systems, domestic water piping, insulation for high tension (high voltage) electrical cables, and baby play mats. It is also used for natural gas and offshore oil applications, chemical transportation, and transportation of sewage and slurries.
Imaging spectroscopyIn imaging spectroscopy (also hyperspectral imaging or spectral imaging) each pixel of an image acquires many bands of light intensity data from the spectrum, instead of just the three bands of the RGB color model. More precisely, it is the simultaneous acquisition of spatially in many spectrally contiguous bands. Some spectral images contain only a few s of a spectral data cube, while others are better thought of as full spectra at every location in the image.
