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Concept
Fluorescence in the life sciences
Summary
Fluorescence 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. 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.
Fluorescence
The principle behind fluorescence is that the fluorescent moiety contains electrons which can absorb a photon and briefly enter an excited state before either dispersing the energy non-radiatively or emitting it as a photon, but with a lower energy, i.e., at a longer wavelength (wavelength and energy are inversely proportional).
The difference in the excitation and emission wavelengths is called the Stokes shift, and the time that an excited electron takes to emit the photon is called a lifetime. The quantum yield is an indicator of the efficiency of the dye (it is the ratio of emitted photons per absorbed photon), and the extinction coefficient is the amount of light that can be absorbed by a fluorophore. Both the quantum yield and extinction coefficient are specific for each fluorophore and multiplied together calculates the brightness of the fluorescent molecule.
fluorophore
Fluorophores can be attached to proteins via specific functional groups, such as:
amino groups (e.g. via succinimide or Isothiocyanate);
carboxyl groups (e.g.
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