A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.
Fluorophores typically contain several combined aromatic groups, or plane 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. But more generally it is covalently bonded to a macromolecule,
serving as a marker for affine or bioactive reagents. Fluorophores are notably used to
stain tissues, cells, or materials in a variety of analytical methods, i.e., fluorescent imaging
and spectroscopy. Fluorescein, by its amine reactive isothiocyanate
derivative FITC, has been one of the most popularized fluorophores. From antibody labeling,
the applications have spread to nucleic acids thanks to, TET,...). Other historically common
fluorophores are derivatives of rhodamine, coumarin, and cyanine. Newer generations of
fluorophores, many of which are proprietary, often perform better than traditional dyes
with comparable excitation and emission.
Fluorescence The fluorophore absorbs light energy of a
specific wavelength and re-emits light at a longer wavelength. The absorbed wavelengths,
energy transfer efficiency, and time before emission depend on both the fluorophore structure
and its chemical environment, as the molecule in its excited state interacts with surrounding
molecules. Wavelengths of maximum absorption and emission are the typical terms used to
refer to a given fluorophore, but the whole spectrum may be important to consider. The
excitation wavelength spectrum may be a very narrow or broader band, or it may be all beyond
a cutoff level. The emission spectrum is usually sharper than the excitation spectrum, and
it is of a longer wavelength and correspondingly lower energy. Excitation energies range from
ultraviolet through the visible spectrum, and emission energies may continue from visible
light into the near infrared region. Main characteristics of fluorophores are :
Maximum excitation and emission wavelength): corresponds to the peak in the excitation
and emission spectra, Extinction Coefficient : links the quantity
of absorbed light, at a given wavelength, to the concentration of fluorophore in solution.
Quantum yield : efficiency of the energy transferred from incident light to emitted
fluorescence Lifetime: duration of the excited state of
a fluorophore before returning to its ground state. It refers to the time taken for a population
of excited fluorophores to decay to 1/e of the original amount.
Stokes shift: difference between the max excitation and max emission wavelengths.
These characteristics drive other properties, including the photobleaching or photoresistance.
Other parameters should be considered, as the polarity of the fluorophore molecule,
the fluorophore size and shape, and other factors can change the behavior of fluorophores.
Fluorophores can also be used to quench the fluorescence of other fluorescent dyes) or
to relay their fluorescence at even longer wavelength
See more on fluorescence principle. Size
Most fluorophores are organic small molecules of 20 - 100 atoms, but there are also much
larger natural fluorophores that are proteins: Green fluorescent protein is 26 kDa and several
phycobiliproteins are ≈240kDa. Fluorescence particles are not considered
as fluorophores The size of the fluorophore might sterically
hinder the tagged molecule, and affect the fluorescence polarity.
Families Fluorophore molecules could be either utilized
alone, or serve as a fluorescent motif of a functional system. Based on molecular complexity
and synthetic methods, fluorophore molecules could be generally classified into four categories:
proteins and peptides, small organic compounds, synthetic oligomers and polymers, and multi-component
systems. fluorescent proteins
GFP, YFP and RFP can be attached to other specific proteins to form a fusion protein,
synthesized in cells after tranfection of a suitable plasmid carrier.
Non-protein organic fluorophores belong to following major chemical families:
Xanthene derivatives: fluorescein, rhodamine, Oregon green, eosin, and Texas red
Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine
Naphthalene derivatives Coumarin derivatives
oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole
Anthracene derivatives: anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange
Pyrene derivatives: cascade blue etc. Oxazine derivatives: Nile red, Nile blue,
cresyl violet, oxazine 170 etc. Acridine derivatives: proflavin, acridine
orange, acridine yellow etc. Arylmethine derivatives: auramine, crystal
violet, malachite green Tetrapyrrole derivatives: porphin, phthalocyanine,
bilirubin These fluorophores fluoresce thanks to delocalized
electrons which can jump a band and stabilize the energy absorbed. Benzene, one of the simplest
aromatic hydrocarbons, for example, is excited at 254 nm and emits at 300 nm. This discriminates
fluorophores from quantum dots, which are fluorescent semiconductor nanoparticles.
They can be attached to protein to specific functional groups, such as - amino groups
- carboxyl groups - thiol - azide). Additionally, various functional groups can
be present to alter its properties, such as solubility, or confer special proprieties,
such as boronic acid which binds to sugars or multiple carboxyl groups to bind to certain
cations. When the dye contains an electron-donating and an electron-accepting group at opposite
ends of the aromatic system, this dye will probably be sensitive to the environment's
polarity, hence called environment-sensitive. Often dyes are used inside cells, which are
impermeable to charged molecules, as a result of this the carboxyl groups are converted
into an ester, which is removed by esterases inside the cells, e.g., fura-2AM and fluorescein-diacetate.
The following dye families are trademark groups, and do not necessarily share structural similarities.
CF dye DRAQ and CyTRAK probes
BODIPY Alexa Fluor
DyLight Fluor Atto and Tracy
FluoProbes Abberior Dyes
DY and MegaStokes Dyes Sulfo Cy dyes
HiLyte Fluor Seta, SeTau and Square Dyes
Quasar and Cal Fluor dyes SureLight Dyes(Columbia Biosciences])
APC, APCXL, RPE, BPE
Examples of frequently encountered fluorophores Reactive and conjugated dyes
Abbreviations: Ex: Excitation wavelength in nanometers
Em: Emission wavelength in nanometers MW: Molecular weight QY: Quantum yield
Nucleic acid dyes Cell function dyes
Fluorescent proteins Abbreviations:
Ex: Excitation wavelength in nanometers Em: Emission wavelength in nanometers
MW: Molecular weight QY: Quantum yield
BR: Brightness: Extinction coefficient * quantum yield / 1000
PS: Photostability: time [sec] to reduce brightness by 50%
Applications Fluorophores have particular importance in
the field of biochemistry and protein studies, e.g., in immunofluorescence but also in cell
analysis, e.g. immunohistochemistry. See detailed techniques and applications in
the article Fluorescence in the life sciences Uses outside the life sciences
Additionally fluorescent dyes find a wide use in industry, going under the name of "neon
colours", such as multi-ton scale usages in textile dyeing and
optical brighteners in laundry detergents advanced cosmetic formulations; safety equipment
and clothing organic light-emitting diodes
fine arts and design synergists for insecticides and experimental
drugs used as a dye in highlighters to give off
a glow-like effect Solar panels
See also Category:Fluorescent dyes
Fluorescence in the life sciences Dark quencher
Fluorescence recovery after photobleaching - an application for quantifying mobility
of molecules in lipid bilayers. References
External links The Database of fluorescent dyes
Table of fluorochromes Fluorescence Tutorials
Fluorescence SpectraViewer - check the compatibility of your fluorophores when designing multicolor
experiments. The Molecular Probes Handbook - a comprehensive
resource for fluorescence technology and its applications.