A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar 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 (when its fluorescence is affected by environmental aspects such as polarity or ions). More generally they are covalently bonded to macromolecules, 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 as fluorescent imaging and spectroscopy.

Fluorescein, via its amine-reactive isothiocyanate derivative fluorescein isothiocyanate (FITC), has been one of the most popular fluorophores. From antibody labeling, the applications have spread to nucleic acids thanks to carboxyfluorescein. Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine. Newer generations of fluorophores, many of which are proprietary, often perform better, being more photostable, brighter, or less pH-sensitive 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, 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 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.

The main characteristics of fluorophores are: Maximum excitation and emission wavelength (expressed in nanometers (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. For quantum dots, prolonged single-molecule microscopy showed that 20-90% of all particles never emit fluorescence. On the other hand, conjugated polymer nanoparticles (Pdots) show almost no dark fraction in their fluorescence. Fluorescent proteins can have a dark fraction from protein misfolding or defective chromophore formation.

These characteristics drive other properties, including photobleaching 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. for polarization fluorescence pattern), 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 wavelengths.

Size (molecular weight) Most fluorophores are organic small molecules of 20–100 atoms (200–1000 Dalton; the molecular weight may be higher depending on grafted modifications and conjugated molecules), but there are also much larger natural fluorophores that are proteins: green fluorescent protein (GFP) is 27 kDa, and several phycobiliproteins (PE, APC...) are ≈240kDa. As of 2020, the smallest known fluorophore was claimed to be 3-hydroxyisonicotinaldehyde, a compound of 14 atoms and only 123 Da.

Fluorescence particles like quantum dots (2–10 nm diameter, 100–100,000 atoms) are also considered 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 (green, yellow, and red, respectively) can be attached to other specific proteins to form a fusion protein, synthesized in cells after transfection 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 Squaraine derivatives and ring-substituted squaraines, including Seta and Square dyes Squaraine rotaxane derivatives: See Tau dyes Naphthalene derivatives (dansyl and prodan 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 Dipyrromethene derivatives: BODIPY, aza-BODIPY

These fluorophores fluoresce due to delocalized electrons which can jump a band and stabilize the energy absorbed. For example, benzene, one of the simplest aromatic hydrocarbons, is excited at 254 nm and emits at 300 nm.Omlc.ogi.edu This discriminates fluorophores from quantum dots, which are fluorescent semiconductor nanoparticles.

They can be attached to proteins to specific functional groups, such as amino groups (active ester, carboxylate, isothiocyanate, hydrazine), carboxyl groups (carbodiimide), thiol (maleimide, acetyl bromide), and organic azide (via click chemistry or non-specifically (glutaraldehyde)).

Additionally, various functional groups can be present to alter their properties, such as solubility, or confer special properties, 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.

The following dye families are trademark 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 (ATTO-TEC GmbH) 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)

Examples of frequently encountered fluorophores

Reactive and conjugated dyes Dye Ex (nm) Em (nm) MW Notes Hydroxycoumarin 325 386 331 Succinimidyl ester Aminocoumarin 350 445 330 Succinimidyl ester Methoxycoumarin 360 410 317 Succinimidyl ester Cascade Blue (375);401 423 596 Hydrazide Pacific Blue 403 455 406 Maleimide Pacific Orange 403 551 3-Hydroxyisonicotinaldehyde 385 525 123 QY 0.15; pH sensitive Lucifer yellow 425 528 NBD 466 539 294 NBD-X R-Phycoerythrin (PE) 480;565 578 240 k PE-Cy5 conjugates 480;565;650 670 aka Cychrome, R670, Tri-Color, Quantum Red PE-Cy7 conjugates 480;565;743 767 Red 613 480;565 613 PE-Texas Red PerCP 490 675 35kDa Peridinin chlorophyll protein TruRed 490,675 695 PerCP-Cy5.5 conjugate FluorX 494 520 587 (GE Healthcare) Fluorescein 495 519 389 FITC; pH sensitive BODIPY-FL 503 512 G-Dye100498524suitable for protein labeling and electrophoresisG-Dye200554575suitable for protein labeling and electrophoresisG-Dye300648663suitable for protein labeling and electrophoresisG-Dye400736760suitable for protein labeling and electrophoresis Cy2 489 506 714 QY 0.12 Cy3 (512);550 570;(615) 767 QY 0.15 Cy3B 558 572;(620) 658 QY 0.67 Cy3.5 581 594;(640) 1102 QY 0.15 Cy5 (625);650 670 792 QY 0.28 Cy5.5 675 694 1272 QY 0.23 Cy7 743 767 818 QY 0.28 TRITC 547 572 444 TRITC X-Rhodamine 570 576 548 XRITC Lissamine Rhodamine B 570 590 Texas Red 589 615 625 Sulfonyl chloride Allophycocyanin (APC) 650 660 104 k APC-Cy7 conjugates 650;755 767 Far Red

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

Nucleic acid dyes Dye Ex (nm) Em (nm) MW Notes Hoechst 33342 343 483 616 AT-selective DAPI 345 455 AT-selective Hoechst 33258 345 478 624 AT-selective SYTOX Blue 431 480 ~400 DNA Chromomycin A3 445 575 CG-selective Mithramycin 445 575 YOYO-1 491 509 1271 Ethidium Bromide 210;285 605 394 in aqueous solutionGelRed290;5205951239Non-toxic substitute for Ethidium Bromide Acridine Orange 503 530/640 DNA/RNA SYTOX Green 504 523 ~600 DNA TOTO-1, TO-PRO-1 509 533 Vital stain, TOTO: Cyanine Dimer TO-PRO: Cyanine Monomer Thiazole Orange 510 530 CyTRAK Orange 520 615 - (Biostatus) (red excitation dark) Propidium Iodide (PI) 536 617 668.4 LDS 751 543;590 712;607 472 DNA (543ex/712em), RNA (590ex/607em) 7-AAD 546 647 7-aminoactinomycin D, CG-selective SYTOX Orange 547 570 ~500 DNA TOTO-3, TO-PRO-3 642 661 DRAQ5 600/647 697 413 (Biostatus) (usable excitation down to 488) DRAQ7 599/644 694 ~700 (Biostatus) (usable excitation down to 488)

Cell function dyes Dye Ex (nm) Em (nm) MW NotesIndo-1 361/330 490/405 1010 AM ester, low/high calcium (Ca2+) Fluo-3 506 526 855 AM ester. pH > 6 Fluo-4 491/494 516 1097 AM ester. pH 7.2 DCFH 505 535 529 2'7'Dichorodihydrofluorescein, oxidized form DHR 505 534 346 Dihydrorhodamine 123, oxidized form, light catalyzes oxidation SNARF 548/579 587/635 pH 6/9

Fluorescent proteins