Fluorescence in the life sciences
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.
This approach allows fluorescent proteins to be used as reporters for any number of biological events, such as sub-cellular localization and expression patterns.
A variant of GFP is naturally found in corals, specifically the Anthozoa, and several mutants have been created to span the visible spectra and fluoresce longer and more stably.
Other proteins are fluorescent but require a fluorophore cofactor, and hence can only be used in vitro; these are often found in plants and algae (phytofluors, phycobiliprotein such as allophycocyanin).
For example, in quantitative PCR, replication of a target nucleic acid sequence is monitored for each cycle by measuring fluorescence intensity.
Within a single reaction, the amplification of multiple nucleic acid sequences can be monitored simultaneously by using fluorophores (e.g. FAM, VIC, Cy5) with distinguishable excitation and emission spectra; this is known as multiplexed qPCR.
10ns) at a lower energy (=higher wavelength), while bioluminescence is biological chemiluminescence, a property where light is generated by a chemical reaction of an enzyme on a substrate.
However the boundary between the fluorescence and phosphorescence is not clean cut as transition metal-ligand complexes, which combine a metal and several organic moieties, have long lifetimes, up to several microseconds (as they display mixed singlet-triplet states).
FRET (Förster resonance energy transfer) is a property in which the energy of the excited electron of one fluorphore, called the donor, is passed on to a nearby acceptor dye, either a dark quencher or another fluorophore, which has an excitation spectrum which overlaps with the emission spectrum of the donor dye resulting in a reduced fluorescence.
This can be used to: Environment-sensitive dyes change their properties (intensity, half-life, and excitation and emission spectra) depending on the polarity (hydrophobicity and charge) of their environments.
Examples include: Indole, Cascade Yellow, prodan, Dansyl, Dapoxyl, NBD, PyMPO, Pyrene and diethylaminocumarin.
Fluorescent moieties emit photons several nanoseconds after absorption following an exponential decay curve, which differs between dyes and depends on the surrounding solvent.
[15] This technique has contributed significantly to the general scientific consensus that mitochondria are physiologically maintained at close to 50 ˚C, more than 10˚C above the rest of the cell.
[16] The inverse relationship between fluorescence and temperature can be explained by the change in the number of atomic collisions in the fluorophore's environment, depending on the kinetic energy.
For example, the thermosensitive fluorophore MTY (MitoTracker Yellow) shows a sudden and drastic drop in fluorescence after the addition of oligomycin (an ATP synthase inhibitor) to the mitochondria of human primary fibroblasts.
Zinc protoporphyrin, formed in developing red blood cells instead of hemoglobin when iron is unavailable or lead is present, has a bright fluorescence and can be used to detect these problems.
Methods of analysis in these fields are also growing, often with nomenclature in the form of acronyms such as: FLIM, FLI, FLIP, CALI, FLIE, FRET, FRAP, FCS, PFRAP, smFRET, FIONA, FRIPS, SHREK, SHRIMP or TIRF.
