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 spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when 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.
In 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. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
Fluorescein is an organic compound and dye. 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.
Fluorescence spectroscopy 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.
A fluorophore 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.
Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 µL per well. Higher density microplates are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.
Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are Rhodamine 6G, Rhodamine 123, and Rhodamine B. They are mainly used to dye paper and inks, but they lack the lightfastness for fabric dying.
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.
DAPI, or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA. It is used extensively in fluorescence microscopy. As DAPI can pass through an intact cell membrane, it can be used to stain both live and fixed cells, though it passes through the membrane less efficiently in live cells and therefore provides a marker for membrane viability.
Texas Red or sulforhodamine 101 acid chloride is a red fluorescent dye, used in histology for staining cell specimens, for sorting cells with fluorescent-activated cell sorting machines, in fluorescence microscopy applications, and in immunohistochemistry. Texas Red fluoresces at about 615 nm, and the peak of its absorption spectrum is at 589 nm. The powder is dark purple. Solutions can be excited by a dye laser tuned to 595-605 nm, or less efficiently a krypton laser at 567 nm. The absorption extinction coefficient at 596 nm is about 85,000 M−1cm−1.
Molecular tagging velocimetry (MTV) is a specific form of flow velocimetry, a technique for determining the velocity of currents in fluids such as air and water. In its simplest form, a single "write" laser beam is shot once through the sample space. Along its path an optically induced chemical process is initiated, resulting in the creation of a new chemical species or in changing the internal energy state of an existing one, so that the molecules struck by the laser beam can be distinguished from the rest of the fluid. Such molecules are said to be "tagged".
Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by intercalation, or RNA via electrostatic attractions. When bound to DNA, acridine orange is very similar spectrally to an organic compound known as fluorescein. Acridine orange and fluorescein have a maximum excitation at 502nm and 525 nm (green). When acridine orange associates with RNA, the fluorescent dye experiences a maximum excitation shift from 525 nm (green) to 460 nm (blue). The shift in maximum excitation also produces a maximum emission of 650 nm (red). Acridine orange is able to withstand low pH environments, allowing the fluorescent dye to penetrate acidic organelles such as lysosomes and phagolysosomes that are membrane-bound organelles essential for acid hydrolysis or for producing products of phagocytosis of apoptotic cells. Acridine orange is used in epifluorescence microscopy and flow cytometry. The ability to penetrate the cell membranes of acidic organelles and cationic properties of acridine orange allows the dye to differentiate between various types of cells. The shift in maximum excitation and emission wavelengths provides a foundation to predict the wavelength at which the cells will stain.
Rhodamine 123 is a chemical compound and a dye. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with instruments called fluorometers. Rhodamine dyes are used extensively in biotechnology applications such as fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy and ELISA. Rhodamine fluorescence can also be used as a measure of membrane polarization in live cell assays both within mitochondria and with bacteria. This use relies on the fact that rhodamine 123 accumulates in membranes in a manner which is dependent on membrane polarization.
6-Carboxyfluorescein (6-FAM) is a fluorescent dye with an absorption wavelength of 495 nm and an emission wavelength of 517 nm. A carboxyfluorescein molecule is a fluorescein molecule with a carboxyl group added. They are commonly used as a tracer agents. It is used in the sequencing of nucleic acids and in the labeling of nucleotides.
A fluorometer or fluorimeter is a device used to measure parameters of visible spectrum fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the amount of specific molecules in a medium. Modern fluorometers are capable of detecting fluorescent molecule concentrations as low as 1 part per trillion.
Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules by means of fluorescence. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. 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. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.
The Qubit fluorometer is a lab instrument developed and distributed by Invitrogen that, among other applications, is used for the quantification of DNA, RNA, and protein.
Microfluorimetry is an adaption of fluorimetry for studying the biochemical and biophysical properties of cells by using microscopy to image cell components tagged with fluorescent molecules. It is a type of microphotometry that gives a quantitative measure of the qualitative nature of fluorescent measurement and therefore, allows for definitive results that would have been previously indiscernible to the naked eye.
Fluorescence polarization immunoassay (FPIA) is a class of in vitro biochemical test used for rapid detection of antibody or antigen in sample. FPIA is a competitive homogenous assay, that consists of a simple prepare and read method, without the requirement of separation or washing steps.
