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Afluorophore (orfluorochrome, similarly to achromophore) is afluorescentchemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combinedaromatic groups, or planar or cyclic molecules with severalπ bonds.^[1]

Fluorophores are sometimes used alone, as atracer in fluids, as adye forstaining of certain structures, as a substrate ofenzymes, or as a probe or indicator (when its fluorescence is affected by environmental aspects such as polarity or ions). More generally they arecovalently bonded tomacromolecules, serving as a markers (or dyes, or tags, or reporters) for affine or bioactive reagents (antibodies, peptides, nucleic acids). Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, such asfluorescent imaging andspectroscopy.

Fluorescein, via itsamine-reactiveisothiocyanate derivativefluorescein isothiocyanate (FITC), has been one of the most popular fluorophores. From antibody labeling, the applications have spread to nucleic acids thanks tocarboxyfluorescein. Other historically common fluorophores are derivatives ofrhodamine (TRITC),coumarin, andcyanine.^[2] Newer generations of fluorophores, many of which are proprietary, often perform better, being more photostable, brighter, or lesspH-sensitive than traditional dyes with comparable excitation and emission.^[3][4]

The fluorophore absorbs light energy of a specific wavelength and re-emits light at a longer wavelength. The absorbedwavelengths,energy transfer efficiency, and time before emission depend on both the fluorophore structure and its chemical environment, since the molecule in its excited state interacts with surrounding molecules. Wavelengths of maximum absorption (≈ excitation) and emission (for example, Absorption/Emission = 485 nm/517 nm) 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 fromultraviolet through thevisible spectrum, and emission energies may continue fromvisible light into thenear infrared region.

The main characteristics of fluorophores are:

• Maximum excitation and emission wavelength (expressed innanometers (nm)): corresponds to the peak in the excitation and emission spectra (usually one peak each).
• Molar absorption coefficient (in mol⁻¹cm⁻¹): 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 (the number of emitted photons per absorbed photons).
• Lifetime (in picoseconds): 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 (≈0.368) of the original amount.
• Stokes shift: the difference between the maximum excitation and maximum emission wavelengths.
• Dark fraction: the proportion of the molecules not active in fluorescence emission. Forquantum dots, prolonged single-molecule microscopy showed that 20-90% of all particles never emit fluorescence.^[5] On the other hand, conjugated polymer nanoparticles (Pdots) show almost no dark fraction in their fluorescence.^[6]Fluorescent proteins can have a dark fraction from protein misfolding or defective chromophore formation.^[7]

These characteristics drive other properties, includingphotobleaching or photoresistance (loss of fluorescence upon continuous light excitation). Other parameters should be considered, as the polarity of the fluorophore molecule, the fluorophore size and shape (i.e. forpolarization fluorescence pattern), and other factors can change the behavior of fluorophores.

Fluorophores can also be used toquench the fluorescence of other fluorescent dyes or torelay their fluorescence at even longer wavelengths.

Most fluorophores are organicsmall molecules of 20–100 atoms (200–1000Dalton; themolecular weight may be higher depending on grafted modifications and conjugated molecules), but there are also much larger natural fluorophores that areproteins:green fluorescent protein (GFP) is 27 kDa, and severalphycobiliproteins (PE, APC...) are ≈240kDa. As of 2020, the smallest known fluorophore was claimed to be3-hydroxyisonicotinaldehyde, a compound of 14 atoms and only 123 Da.^[8]

Fluorescence particles likequantum dots (2–10 nm diameter, 100–100,000 atoms) are also considered fluorophores.^[9]

The size of the fluorophore mightsterically hinder the tagged molecule and affect the fluorescence polarity.

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.^[10][11]

Fluorescent proteins GFP, YFP, and RFP (green, yellow, and red, respectively) can be attached to other specific proteins to form afusion protein, synthesized in cells aftertransfection of a suitableplasmid carrier.

Non-protein organic fluorophores belong to following major chemical families:

• Xanthene derivatives:fluorescein,rhodamine,Oregon green,eosin, andTexas red
• Cyanine derivatives: cyanine,indocarbocyanine,oxacarbocyanine,thiacarbocyanine, andmerocyanine
• Squaraine derivatives and ring-substituted squaraines, including Seta and Square dyes
• Squaraine rotaxane derivatives: See Tau dyes
• Naphthalene derivatives (dansyl andprodan derivatives)
• Coumarin derivatives
• Oxadiazole derivatives:pyridyloxazole,nitrobenzoxadiazole, andbenzoxadiazole
• 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
• Dipyrromethene derivatives:BODIPY,aza-BODIPY

These fluorophores fluoresce due todelocalized electrons which can jump aband and stabilize the energy absorbed. For example,benzene, one of the simplest aromatic hydrocarbons, is excited at 254 nm and emits at 300 nm.^[12] This discriminates fluorophores from quantum dots, which are fluorescent semiconductornanoparticles.

They can be attached to proteins to specific functional groups, such asamino groups (active ester,carboxylate,isothiocyanate,hydrazine),carboxyl groups (carbodiimide),thiol (maleimide,acetyl bromide), andorganic azide (viaclick chemistry or non-specifically (glutaraldehyde)).

Additionally, various functional groups can be present to alter their properties, such as solubility, or confer special properties, such asboronic acid which binds to sugars or multiplecarboxyl 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 andfluorescein-diacetate.

The following dye families aretrademark groups, and do not necessarily share structural similarities.

• CF dye (Biotium)
• DRAQ and CyTRAK probes (BioStatus)
• BODIPY (Invitrogen)
• EverFluor (Setareh Biotech)
• Alexa Fluor (Invitrogen)
• Bella Fluor (Setareh Biotech)
• DyLight Fluor (Thermo Scientific, Pierce)
• Atto and Tracy (Sigma Aldrich)
• FluoProbes (Interchim)
• Abberior Dyes (Abberior)
• DY and MegaStokes Dyes (Dyomics)
• Sulfo Cy dyes (Cyandye)
• HiLyte Fluor (AnaSpec)
• Seta, SeTau and Square Dyes (SETA BioMedicals)
• Quasar and Cal Fluor dyes (Biosearch Technologies)
• SureLight Dyes (APC, RPEPerCP,Phycobilisomes) (Columbia Biosciences)
• APC, APCXL, RPE, BPE (Phyco-Biotech, Greensea, Prozyme, Flogen)
• Vio Dyes (Miltenyi Biotec)

Abbreviations:

• Ex (nm): Excitation wavelength innanometers
• Em (nm): Emission wavelength in nanometers
• MW:Molecular weight
• QY:Quantum yield

StayGold andmStayGold are advanced fluorescent proteins that have significantly contributed to the field of live-cell imaging. StayGold, known for its high photostability and brightness, was originally designed as a dimeric fluorescent protein, which, while effective, posed challenges related to the aggregation and labelling accuracy.^[15] To address these limitations, mStayGold was engineered as a monomeric variant, enhancing its utility in precise protein labeling. mStayGold exhibits superior photostability, maintaining fluorescence under high irradiance conditions and demonstrates increased brightness compared to its former variant StayGold. Additionally, it matures faster, allowing for quicker imaging post-transfection. These advancements make mStayGold a versatile tool for a variety of applications, including single molecule tracking and high resolution imaging of dynamic cellular processes, thereby expanding the capabilities of fluorescent protein in biological research.^[16]

Abbreviations:

• Ex (nm): Excitation wavelength innanometers
• Em (nm): Emission wavelength in nanometers
• MW:Molecular weight
• QY:Quantum yield
• BR: Brightness: Molar absorption coefficient * quantum yield / 1000
• PS:Photostability: time [sec] to reduce brightness by 50%

Fluorophores have particular importance in the field ofbiochemistry andprotein studies, for example, inimmunofluorescence, cell analysis,^[17]immunohistochemistry,^[3][18] andsmall molecule sensors.^[19][20]

Fluorescent dyes find a wide use in industry, going under the name of "neon colors", such as:

• Multi-ton scale usages in textile dyeing and optical brighteners inlaundry detergents
• Advancedcosmetic formulations
• Safety equipment and clothing
• Organic light-emitting diodes (OLEDs)
• Fine arts and design (posters and paintings)
• Synergists for insecticides and experimental drugs
• Dyes inhighlighters to give off a glow-like effect
• Solar panels to collect more light / wavelengths
• Fluorescent sea dye is used to help airbornesearch and rescue teams locate objects in the water

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

• The Database of fluorescent dyes
• Table of fluorochromes
• The Molecular Probes Handbook - a comprehensive resource for fluorescence technology and its applications.

• Dyes
• Luminescence

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• Short description matches Wikidata
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• All pages needing cleanup
• Wikipedia list cleanup from February 2016