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.
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.
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.
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.
Emission spectrumThe emission spectrum of a chemical element or chemical compound is the spectrum of frequencies of electromagnetic radiation emitted due to an electron making a transition from a high energy state to a lower energy state. The photon energy of the emitted photon is equal to the energy difference between the two states. There are many possible electron transitions for each atom, and each transition has a specific energy difference. This collection of different transitions, leading to different radiated wavelengths, make up an emission spectrum.
EnergyIn physics, energy () is the quantitative property that is transferred to a body or to a physical system, recognizable in the performance of work and in the form of heat and light. Energy is a conserved quantity—the law of conservation of energy states that energy can be converted in form, but not created or destroyed. The unit of measurement for energy in the International System of Units (SI) is the joule (J).
Excited stateIn quantum mechanics, an excited state of a system (such as an atom, molecule or nucleus) is any quantum state of the system that has a higher energy than the ground state (that is, more energy than the absolute minimum). Excitation refers to an increase in energy level above a chosen starting point, usually the ground state, but sometimes an already excited state. The temperature of a group of particles is indicative of the level of excitation (with the notable exception of systems that exhibit negative temperature).
Rotational symmetryRotational symmetry, also known as radial symmetry in geometry, is the property a shape has when it looks the same after some rotation by a partial turn. An object's degree of rotational symmetry is the number of distinct orientations in which it looks exactly the same for each rotation. Certain geometric objects are partially symmetrical when rotated at certain angles such as squares rotated 90°, however the only geometric objects that are fully rotationally symmetric at any angle are spheres, circles and other spheroids.
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.
Absorption spectroscopyAbsorption spectroscopy refers to spectroscopic techniques that measure the absorption of electromagnetic radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of the absorption varies as a function of frequency, and this variation is the absorption spectrum. Absorption spectroscopy is performed across the electromagnetic spectrum.
