Fluorophore

For uses and theory of fluorescence, see Fluorescence in the life sciences.

A fluorophore is a fluorochrome (or fluorescent chromophore) covalently bonded to a macromolecule and used to stain tissues, cells, or materials for fluorescent imaging and spectroscopy. The fluorphore absorbs light energy of a specific wavelength and re-emits energy at a longer wavelength. The wavelength, amount, and time before emission of the emitted energy depend on both the fluorophore and its chemical environment as the molecule in its excited state interacts with surrounding molecules. Flourophores are selected for both the energy required to excite the molecule or structure of interest and the molecule's emission energy. The excitation energy may be a very narrow or broader band of energy, or it may be all energies beyond a cutoff level. The emission energy is usually more specific than the excitation energy, and it is of a longer wavelength or lower energy. Excitation energies range from ultraviolet through the visible spectrum, and emission energies may continue from visible light into the near infrared region. This technology has particular importance in the field of biochemistry and protein studies, e.g., 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, many of which are proprietary, often perform better (more photostable, brighter, and/or less pH-sensitive) than traditional dyes with comparable excitation and emission.[1][3]

Contents

Size

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

Families

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

These fluorophores are either quantum dots or small molecules. The former are 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.[4]

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:

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

A few examples of commonly used dyes:

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
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

CyDyes

Probe Ex (nm) Em (nm) MW Quantum yield
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

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
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
mCherry 587 610 0.22 16 96 monomer
HcRed1 588 618 ~52k 0.03 0.6 dimer, (Clontech)
Katusha 588 635 23 dimer
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%

Uses outside the life sciences

Additionally fluorescent dyes find a wide use in industry, going under the name of "neon colours", such as

See also

References

  1. ^ a b Tsien RY, Waggoner A (1995). "Fluorophores for confocal microscopy". In Pawley JB. Handbook of biological confocal microscopy. New York: Plenum Press. pp. 267–74. ISBN 0-306-44826-2. http://books.google.com/books?id=16Ft5k8RC-AC&pg=PA267. Retrieved 2008-12-13. 
  2. ^ Rietdorf J (2005). Microscopic Techniques. Advances in Biochemical Engineering / Biotechnology. Berlin: Springer. pp. 246–9. ISBN 3-540-23698-8. http://books.google.com/books?id=h9F_RGrIoicC&pg=PA247. Retrieved 2008-12-13. 
  3. ^ Lakowicz, JR (2006). Principles of fluorescence spectroscopy (3rd ed.). Springer. pp. 954. ISBN 978-0387312781. 
  4. ^ Omlc.ogi.edu
  5. ^ Pingu.salk.edu

External links