Fluorophore

A fluorophore-labeled human cell.

A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. This technology has particular importance in the field of biochemistry and protein studies, eg. in immunofluorescence and immunohistochemistry.^[1]

Fluorescein isothiocyanate (FITC), a reactive derivative of fluorescein, has been one of the most common fluorophores chemically attached to other, non-fluorescent molecules to create new fluorescent molecules for a variety of applications. Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine.^[2] Newer generations of fluorophores such as the CF Dyes, HiLyte Fluors, the Alexa Fluors and the DyLight Fluors are generally more photostable, brighter, and less pH-sensitive than other standard dyes of comparable excitation and emission.^{[1] [3] [4]}

Size

The size of the fluorophore might sterically hinder the tagged molecule:

• quantum dot: 2-10 nm (diameter), 100-100,000 atoms
• protein: Green fluorescent protein (GFP) 26 kDa
• small molecule: fluorescein: about 20 atoms

Families

Several fluorescent protein exist and are attached to a specific protein in a plasmid to form a fusion protein. Fluorophores can be attached to protein to specific functional groups, such as

• amino groups (succinimide, Isothiocyanate, hydrazine)
• carboxyl groups (Carbodiimide)
• thiol (maleimide, acetyl bromide)
• azide (via click chemistry)

or non-specificately (Glutaraldehyde).

These fluorophores are either quantum dots or small molecules. The former a fluorescent semiconductor nanoparticles. The latter molecules which fluoresce thanks to delocalized electrons which can jump a band and stabilize the energy absorbed, hence all fluorophores are aromatic or conjugated. Benzene, one of the simplest aromatic hydrocarbons, for example, is excited at 254 nm and emits at 300 nm ^[5].
Additionally, various functional groups can be present to alter its proprieties, 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 (solvatochromic), 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.

Common dye families are:

• Xanthene derivatives: Fluorescein, Rhodamine, Oregon green, eosin, texas red etc.
• Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine
• Naphtalene derivatives (Dansyl and Prodan derivatives)
• Coumarin derivatives
• oxadiazole derivatives: Pyridyloxazole, Nitrobenzoxadiazole and Benzoxadiazole
• Pyrene derivatives: Cascade blue etc.
• BODIPY (invitrogen)
• 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
• CF dye (Biotium)
• Alexa Fluor (invitrogen) - a trade grouping and do not share structures
• Atto and Tracy (sigma) - a trade grouping and do not share structures
• Tetrapyrrole derivatives: porphin, phtalocyanine, bilirubin
• others: cascade yellow, azure B, acridine orange, DAPI, Hoechst 33258, lucifer yellow, Piroxicam, quinine and anthraqinone, squarylium, oligophenylenes, etc.

A few examples of commonly used dyes:

Bovine Pulmonary Artery Endothelial cell nuclei stained blue with DAPI, mitochondria stained red with MitoTracker Red CMXRos, and F-actin stained green with Alexa Fluor 488 phalloidin and imaged on a fluorescent microscope.

Reactive and conjugated probes

Alexa Fluor dyes [antibody conjugates] (Molecular Probes)

Cy Dyes (GE Heathcare)

Nucleic acid probes

Cell function probes

Fluorescent proteins

^[6]

Other uses

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 (OLED)
• fine arts and design (posters and paintings)
• synergists for insecticides and experimental drugs

See also

• Dark quencher
• Category:Fluorescent dyes
• Fluorescence recovery after photobleaching (FRAP) - an application for quantifying mobility of molecules in lipid bilayers.
• Fluorescence in the life sciences

References

External links

• The Database of fluorescent dyes
• Table of fluorochromes