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Fluorophore

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For uses and theory of fluorescence, see Fluorescence in the life sciences.
A fluorophore-labeled human cell.

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 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 (when its fluorescence is affected by environment such as polarity, ions,...). But more generally it is covalently bonded to a macromolecule, serving as a marker (or dye, or tag, or reporter) 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, 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 (FAM(Carboxyfluorescein), TET,...). Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine.[1] Newer generations of fluorophores, many of which are proprietary, often perform better (more photostable, brighter, and/or less pH-sensitive) than traditional dyes with comparable excitation and emission.[2][3]

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. Maximum wavelengths of 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 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 (expressed in nanometers (nm)): corresponds to the peak in the excitation and emission spectra (usually one peak each),
  • Extinction Coefficient (or molar absorption, 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 (= 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: difference between the max excitation and max emission wavelengths.

These characteristics drive other properties, including the 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 (see article Quenching (fluorescence)) or to relay their fluorescence at even longer wavelength (see article FRET)

See more on fluorescence principle.

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 they are also much larger natural proteins: Green fluorescent protein (GFP) is 26 kDa and several phycobiliproteins(PE, APC...) are ≈240kDa .

Fluorescence particles are not considered as fluorophores (quantum dot: 2-10 nm diameter, 100-100,000 atoms)

The size of the fluorophore might sterically hinder the tagged molecule, and affect the fluorescence polarity.

Families

GFP (green), YFP (yellow) and RFP (red) 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 (dansyl and prodan derivatives) - Coumarin derivatives - oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole - 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, phtalocyanine, 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.[4] This discrimates Fluorophores from quantum dots, which are fluorescent semiconductor nanoparticles.

  • They can be attached to protein to specific functional groups, such as

- amino groups (Active ester, Carboxylate, Isothiocyanate, hydrazine) - carboxyl groups (carbodiimide) - thiol (maleimide, acetyl bromide) - azide (via click chemistry or non-specifically (glutaraldehyde)).

Additionally, various functional groups can be present to alter its proprerties, 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.

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

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

Probe 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
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 Peridinin chlorphyll 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
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 1128 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 probes

Probe 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 493 620 394
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
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 647 681/697 413 (Biostatus) (usable excitation down to 488)

Cell function probes

Probe Ex (nm) Em (nm) MW Notes
Indo-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[5]

Probe Ex (nm) Em (nm) MW QY BR PS Notes
Y66H 360 442
Y66F 360 508
EBFP 380 440 0.18 0.27 monomer
EBFP2 383 448 20 monomer
Azurite 383 447 15 monomer
GFPuv 385 508
T-Sapphire 399 511 0.60 26 25 weak dimer
Cerulean 433 475 0.62 27 36 weak dimer
mCFP 433 475 0.40 13 64 monomer
ECFP 434 477 0.15 3
CyPet 435 477 0.51 18 59 weak dimer
Y66W 436 485
mKeima-Red 440 620 0.24 3 monomer (MBL)
TagCFP 458 480 29 dimer (Evrogen)
AmCyan1 458 489 0.75 29 tetramer, (Clontech)
mTFP1 462 492 54 dimer
S65A 471 504
Midoriishi Cyan 472 495 0.9 25 dimer (MBL)
Wild Type GFP 396,475 508 26k 0.77
S65C 479 507
TurboGFP 482 502 26 k 0.53 37 dimer, (Evrogen)
TagGFP 482 505 34 monomer (Evrogen)
S65L 484 510
Emerald 487 509 0.68 39 0.69 weak dimer, (Invitrogen)
S65T 488 511
EGFP 488 507 26k 0.60 34 174 weak dimer, (Clontech)
Azami Green 492 505 0.74 41 monomer (MBL)
ZsGreen1 493 505 105k 0.91 40 tetramer, (Clontech)
TagYFP 508 524 47 monomer (Evrogen)
EYFP 514 527 26k 0.61 51 60 weak dimer, (Clontech)
Topaz 514 527 57 monomer
Venus 515 528 0.57 53 15 weak dimer
mCitrine 516 529 0.76 59 49 monomer
YPet 517 530 0.77 80 49 weak dimer
TurboYFP 525 538 26 k 0.53 55.7 dimer, (Evrogen)
ZsYellow1 529 539 0.65 13 tetramer, (Clontech)
Kusabira Orange 548 559 0.60 31 monomer (MBL)
mOrange 548 562 0.69 49 9 monomer
Allophycocyanin (APC) 652 657.5 105 kDa 0.68 heterodimer, crosslinked[6]
mKO 548 559 0.60 31 122 monomer
TurboRFP 553 574 26 k 0.67 62 dimer, (Evrogen)
tdTomato 554 581 0.69 95 98 tandem dimer
TagRFP 555 584 50 monomer (Evrogen)
DsRed monomer 556 586 ~28k 0.1 3.5 16 monomer, (Clontech)
DsRed2 ("RFP") 563 582 ~110k 0.55 24 (Clontech)
mStrawberry 574 596 0.29 26 15 monomer
TurboFP602 574 602 26 k 0.35 26 dimer, (Evrogen)
AsRed2 576 592 ~110k 0.21 13 tetramer, (Clontech)
mRFP1 584 607 ~30k 0.25 monomer, (Tsien lab)
J-Red 584 610 0.20 8.8 13 dimer
R-phycoerythrin (RPE) 565 >498 573 250 kDa 0.84 heterotrimer[6]
B-phycoerythrin (BPE) 545 572 240 kDa 0.98 heterotrimer[6]
mCherry 587 610 0.22 16 96 monomer
HcRed1 588 618 ~52k 0.03 0.6 dimer, (Clontech)
Katusha 588 635 23 dimer
P3 614 662 ~10,000 kDa phycobilisome complex[6]
Peridinin Chlorophyll (PerCP) 483 676 35 kDa trimer [6]
mKate (TagFP635) 588 635 15 monomer (Evrogen)
TurboFP635 588 635 26 k 0.34 22 dimer, (Evrogen)
mPlum 590 649 51.4 k 0.10 4.1 53
mRaspberry 598 625 0.15 13 monomer, faster photobleach than mPlum

Abbreviations:
Ex (nm): Excitation wavelength in nanometers
Em (nm): 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.[2]

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 (OLED)
  • fine arts and design (posters and paintings)
  • synergists for insecticides and experimental drugs
  • used as a dye in highlighters to give off a glow-like effect
  • Solar panels (collect more light / wavelengths)

See also

References

  1. ^ Rietdorf J (2005). Microscopic Techniques. Advances in Biochemical Engineering / Biotechnology. Berlin: Springer. pp. 246–9. ISBN 3-540-23698-8. Retrieved 2008-12-13.
  2. ^ a b Tsien RY, Waggoner A (1995). "Fluorophores for confocal microscopy". In Pawley JB (ed.). Handbook of biological confocal microscopy. New York: Plenum Press. pp. 267–74. ISBN 0-306-44826-2. Retrieved 2008-12-13.
  3. ^ Lakowicz, JR (2006). Principles of fluorescence spectroscopy (3rd ed.). Springer. p. 954. ISBN 978-0-387-31278-1.
  4. ^ Omlc.ogi.edu
  5. ^ Pingu.salk.edu
  6. ^ a b c d e Columbia Biosciences
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