出典(authority):フリー百科事典『ウィキペディア(Wikipedia)』「2014/12/19 05:24:56」(JST)
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]
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. 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 from ultraviolet through the visible spectrum, and emission energies may continue from visible light into the near infrared region.
Main characteristics of fluorophores are :
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.
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 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.
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. [4]
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:
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.[5] This discriminates 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 properties, 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.
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 | ||
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 | ||
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 |
SeTau-647 | 649 | 695 | 1461 | QY 0.61; Lifetime 3.2 ns |
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
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 | 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 | ||
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) |
Dye | 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 |
Dye | Ex (nm) | Em (nm) | MW | QY | BR | PS | Notes |
---|---|---|---|---|---|---|---|
GFP (Y66H mutation) | 360 | 442 | |||||
GFP (Y66F mutation) | 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 | |
mTurquoise2 | 434 | 474 | 0.93 | 28 | monomer | ||
ECFP | 434 | 477 | 0.15 | 3 | |||
CyPet | 435 | 477 | 0.51 | 18 | 59 | weak dimer | |
GFP (Y66W mutation) | 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 | |||
GFP (S65A mutation) | 471 | 504 | |||||
Midoriishi Cyan | 472 | 495 | 0.9 | 25 | dimer (MBL) | ||
Wild Type GFP | 396,475 | 508 | 26k | 0.77 | |||
GFP (S65C mutation) | 479 | 507 | |||||
TurboGFP | 482 | 502 | 26 k | 0.53 | 37 | dimer, (Evrogen) | |
TagGFP | 482 | 505 | 34 | monomer (Evrogen) | |||
GFP (S65L mutation) | 484 | 510 | |||||
Emerald | 487 | 509 | 0.68 | 39 | 0.69 | weak dimer, (Invitrogen) | |
GFP (S65T mutation) | 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[7] | ||
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[7] | ||
B-phycoerythrin (BPE) | 545 | 572 | 240 kDa | 0.98 | heterotrimer[7] | ||
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[7] | |||
Peridinin Chlorophyll (PerCP) | 483 | 676 | 35 kDa | trimer[7] | |||
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%
Fluorophores have particular importance in the field of biochemistry and protein studies, e.g., in immunofluorescence but also in cell analysis, e.g. immunohistochemistry.[8] [2]
See detailed techniques and applications in the article Fluorescence in the life sciences
Additionally fluorescent dyes find a wide use in industry, going under the name of "neon colours", such as
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リンク元 | 「fluorochrome」「fluorescent probe」「fluorogenic substrate」 |
関連記事 | 「dye」「fluorescent」「dying」 |
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