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== See also ==
== See also ==
{{further|http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook.html}} <!-- This is not an add, Invitrogen has a kinda monopoly so has the best litterature -->


* [[Dark quencher]]
* [[Dark quencher]]

Revision as of 14:21, 21 July 2009

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 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:

Families

Fluorophores can be attached to protein to specific functional groups, such as

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 most fluorophores are aromatic, a propriety that can arise is that when polar molecules stabilize one resonance structure more over the other the dye is sensitive to the environment's polarity (solvatochromic), hence called environmentally sensitive.

Common dye families are:

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 PharRed

Alexa Fluor dyes [antibody conjugates] (Molecular Probes)

Probe Ex (nm) Em (nm) MW Quencher
Alexa Fluor 350 343 442 410
Alexa Fluor 405 401 421 1028
Alexa Fluor 430 434 540 702
Alexa Fluor 488 499 519 643 QY 0.92
Alexa Fluor 500 503 525 700
Alexa Fluor 514 517 542 714
Alexa Fluor 532 530 555 724 QY 0.61
Alexa Fluor 546 561 572 1079 QY 0.79
Alexa Fluor 555 553 568 1250 QY 0.1
Alexa Fluor 568 579 603 792 QY 0.69
Alexa Fluor 594 591 618 820 QY 0.66
Alexa Fluor 610 610 629 1285
Alexa Fluor 633 632 648 1200
Alexa Fluor 647 652 668 1300 QY 0.33
Alexa Fluor 660 663 691 1100
Alexa Fluor 680 680 702 1150
Alexa Fluor 700 696 719 1400
Alexa Fluor 750 752 776 1300
Alexa Fluor 790 782 804 1750

Cy Dyes (GE Heathcare)

Probe Ex (nm) Em (nm) MW Quencher
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 Ca++,
Fluo-3 506 526 855 AM ester. pH > 6
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

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 1.65 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 0.10 4.1 53
mRaspberry 598 625 0.15 13 monomer; faster photobleach than mPlum

[5]

See also

Further information: http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook.html

References

  1. ^ a b Tsien RY, Waggoner Aeditor=Pawley JB (1995). "Fluorophores for confocal microscopy". Handbook of biological confocal microscopy. New York: Plenum Press. pp. 267–74. ISBN 0-306-44826-2. 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. Retrieved 2008-12-13.
  3. ^ Lakowicz, J.R., Principles of fluorescence spectroscopy. 3rd ed. 2006, New York: Springer. xxvi, 954 p.
  4. ^ http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Introduction-to-Fluorescence-Techniques.html
  5. ^ http://pingu.salk.edu/flow/fluo.html
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