StainingStaining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissues), in cytology (microscopic study of cells), and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells), or organelles within individual cells.
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).
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.
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.
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.
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.
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.
FluoresceinFluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications. The color of its aqueous solutions is green by reflection and orange by transmission (its spectral properties are dependent on pH of the solution), as can be noticed in bubble levels, for example, in which fluorescein is added as a colorant to the alcohol filling the tube in order to increase the visibility of the air bubble contained within.
Phosphotungstic acidPhosphotungstic acid (PTA) or tungstophosphoric acid (TPA), is a heteropoly acid with the chemical formula . It forms hydrates . It is normally isolated as the n = 24 hydrate but can be desiccated to the hexahydrate (n = 6). EPTA is the name of ethanolic phosphotungstic acid, its alcohol solution used in biology. It has the appearance of small, colorless-grayish or slightly yellow-green crystals, with melting point 89 °C (24 hydrate). It is odorless and soluble in water (200 g/100 ml).
PeriodatePeriodate pəˈraɪ.ədeɪt is an anion composed of iodine and oxygen. It is one of a number of oxyanions of iodine and is the highest in the series, with iodine existing in oxidation state +7. Unlike other perhalogenates, such as perchlorate, it can exist in two forms: metaperiodate IO4− and orthoperiodate IO65−. In this regard it is comparable to the tellurate ion from the adjacent group. It can combine with a number of counter ions to form periodates, which may also be regarded as the salts of periodic acid.
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.
Keggin structureKeggin structure is the best known structural form for heteropoly acids. It is the structural form of α-Keggin anions, which have a general formula of , where X is the heteroatom (most commonly are pentavalent phosphorus PV, tetravalent silicon SiIV, or trivalent boron BIII), M is the addendum atom (most common are molybdenum Mo and tungsten W), and O represents oxygen. The structure self-assembles in acidic aqueous solution and is the most stable structure of polyoxometalate catalysts.
HexamethylenetetramineHexamethylenetetramine, also known as methenamine, hexamine, or urotropin, is a heterocyclic organic compound with the formula (CH2)6N4. This white crystalline compound is highly soluble in water and polar organic solvents. It has a cage-like structure similar to adamantane. It is useful in the synthesis of other organic compounds, including plastics, pharmaceuticals, and rubber additives. It sublimes in vacuum at 280 °C. Hexamethylenetetramine was discovered by Aleksandr Butlerov in 1859.
Malachite greenMalachite green is an organic compound that is used as a dyestuff and controversially as an antimicrobial in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper. Despite its name the dye is not prepared from the mineral malachite; the name just comes from the similarity of color. Malachite green is classified in the dyestuff industry as a triarylmethane dye and also using in pigment industry.
Propidium iodidePropidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a fluorescent excitation maximum of 493 nm (blue-green), and an emission maximum of 636 nm (red). After binding DNA, the quantum yield of PI is enhanced 20-30 fold, and the excitation/emission maximum of PI is shifted to 535 nm (green) / 617 nm (orange-red).
Methylene blueMethylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. As a medication, it is mainly used to treat methemoglobinemia by converting/chemically reducing the ferric iron in hemoglobin to ferrous iron. Specifically, it is used to treat methemoglobin levels that are greater than 30% or in which there are symptoms despite oxygen therapy. It has previously been used for treating cyanide poisoning and urinary tract infections, but this use is no longer recommended.
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.
Quenching (fluorescence)In chemistry, quenching refers to any process which decreases the fluorescent intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisions. As a consequence, quenching is often heavily dependent on pressure and temperature. Molecular oxygen, iodine ions and acrylamide are common chemical quenchers. The chloride ion is a well known quencher for quinine fluorescence.
Methylene (compound)Methylene (IUPAC name: Methylidene, also called carbene or methene) is an organic compound with the chemical formula CH2 (also written [CH2]). It is a colourless gas that fluoresces in the mid-infrared range, and only persists in dilution, or as an adduct. Methylene is the simplest carbene. It is usually detected only at very low temperatures, or as a short-lived intermediate in chemical reactions. The trivial name carbene is the preferred IUPAC name.
EosinEosin is the name of several fluorescent acidic compounds which bind to and form salts with basic, or eosinophilic, compounds like proteins containing amino acid residues such as arginine and lysine, and stains them dark red or pink as a result of the actions of bromine on eosin. In addition to staining proteins in the cytoplasm, it can be used to stain collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic.
