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Fluorescence Excitation and Emission

Most fluorescent dyes are heterocyclic or polyaromatic hydrocarbons. Light is absorbed by the dye, which leads to an excited state of the fluorophore that has a lifetime of 10-9 - 10-8 seconds (nanoseconds). All the dyes discussed below absorb and emit in the visible or near infrared regions. It is an often forgotten but useful fact the many dyes can also be effectively excited from a UV source. While in the excited state, the fluorophore can undergo conformational changes or interact with the environment such that energy is dissipated (as heat) or transferred to neighboring molecules (energy transfer - see below). When the fluorophore returns from the first excited state to the ground state, light is emitted at lower energy (longer wavelength) than was originally captured. The wavelength difference between the light captured (absorbance/excitation) and released (emission) is called the Stokes shift. A large Stokes shift is desirable as it simplifies the technical aspects of achieving concurrent excitation with emission detection and also decreased interfering background. In summary, optical energy captured by a fluorophore has three possible fates:

  1. emitted as light (fluorescence),
  2. resonance energy transfer (to another group), or
  3. dissipated as heat

The energy capture efficiency of a fluorescent dye is expressed as the extinction coefficient, ε, and usually ranges from 10,000 to 250,000 cm-1M-1. The emission efficiency is expressed as the quantum yield, QY. Quantum yield is defined as the ratio of the number of photons emitted to the number of photons absorbed by a fluorophore. Since the fluorescence of many fluorophores are strongly influenced by local environment, the resulting quantum yields vary as well (and are therefore not usually reported as a "constant" like the extinction coefficient). An ideal fluorophore would have a high extinction coefficient and a quantum yield close to unity. However, other factors may play significant roles as well. For example, fluorescein has an extinction coefficient of 78,000 with a high QY. While Tamra (carboxytetramethylrhodamine) has an extinction coefficient of 91,000 with a quantum yield about that of fluorescein, its fluorescence will often appear " brighter" than fluorescein due to the susceptibility of fluorescein photobleaching. In addition, fluorescein is partially quenched after conjugation to proteins or nucleic acids which results in a 50% drop in quantum yield.

The maximal absorption and emission wavelengths and the extinction coefficients of the most common fluorophores are listed below:

Table 1.
The maximal absorption wavelength, extinction coefficient, and the maximal emission wavelength of common fluorophores in the form of activated NHS-ester with a linker arm.

Dye Abmax
(nm)
Extinction Coefficient
(Lmole-1cm-1)
Emmax
(nm)
Acridine 362 11,000 462
AMCA 353 19,000 442
BODIPY FL-Br2 531 75,000 545
BODIPY 530/550 534 77,000 554
BODIPY TMR 544 56,000 570
BODIPY 558/568 558 97,000 569
BODIPY 564/570 563 142,000 569
BODIPY 576/589 575 83,000 588
BODIPY 581/591 581 136,000 591
BODIPY TR 588 68,000 616
BODIPY 630/650 625 101,000 640
BODIPY 650/665 646 102,000 660
Cascade Blue 396 29,000 410
Cy2 489 150,000 506
Cy3 552 150,000 570
Cy3.5 581 150,000 596
Cy5 643 250,000 667
Cy5.5 675 250,000 694
Cy7 743 250,000 767
Dabcyl 453 32,000 none
Edans 335 5,900 493
Eosin 521 95,000 544
Erythrosin 529 90,000 553
Fluorescein 492 78,000 520
6-Fam 494 83,000 518
Tet 521 - 536
Joe 520 71,000 548
Hex 535 - 556
LightCycler 640 625 110,000 640
LightCycler 705 685 - 705
NBD 465 22,000 535
Oregon Green 488 492 88,000 517
500 499 78,000 519
514 506 85,000 526
Rhodamine 6G 524 102,000 550
Rhodamine Green 504 78,000 532
Rhodamine Red 560 129,000 580
Rhodol Green 496 63,000 523
Tamra 565 91,000 580
Rox 585 82,000 605
Texas Red 583 116,000 603
NED 546 - 575
VIC 538 - 554

Photobleaching is the irreversible destruction of a fluorophore in the excited state. Different fluorophores have different rates of photobleaching. The best solution to photobleaching is to use a maximally sensitive detection system with lower intensity illumination. High intensity illumination accelerates photobleaching. Use of a high sensitivity detector with broadband pass filters will improve performance of a rapidly photobleaching dye such as fluorescein. Alternatively, a more photo stable dye with similar absorbance/emission spectra can be substituted, such as Oregon Green for fluorescein.

The spectral properties of some fluorescent dyes are pH sensitive. For example, fluorescein has a pKa of ~6.4 and its quantum yield and fluorescence intensity rapidly falls off as pH drops below 7. Rhodamine based dyes, such as Rox or Tamra, are relatively pH insensitive. Cy5 is physically unstable in acid conditions and should be stored and used in buffers above pH 7.

UV excitation of dyes such as fluorescein is not as efficient as stimulation with light at the Absmax in the visible range. However, UV excitation offers a convenient method of exciting a series of different fluorescence imaging systems where a simple UV source and one filter can be used for photo documentation of a wide variety of fluorophores.
Fluorescence Energy Transfer and Quenching

Following excitation, energy can dissipate from a fluorescent dye to neighboring molecules. Some energy is lost to solvent. Nearby heterocyclic ring structures, as are present when the dye is conjugated to a nucleic acid or protein, will absorb some of this energy and "quench" the dye. Fluorescence resonance energy transfer (FRET) occurs when energy passed from one fluorophore to another without emission of light. FRET requires that the two groups be in close proximity, usually 10-100 , and energy transfer efficiency decreases by the sixth power with increasing spatial separation. Further, the absorption spectrum of the acceptor must overlap the emission spectrum of the donor and the donor and acceptor dipole orientations must be approximately parallel. In one commonly used donor/acceptor pair, fluorescein is efficiently quenched by Tamra if the two groups are within 55 of each other. Diffusional energy transfer can also occur and requires the interacting groups to either be in contact or very close proximity and occurs through a mechanism distinct from FRET. For more information on FRET, CLICK HERE.

FRET is exploited today to suppress background and improve convenience in some assays. For example, in the TaqMan 5'-nuclease assay, two fluorescent dyes (such as Fam and Tamra) are brought into physical proximity by direct conjugation at opposite ends of a short oligo and interact. When the high-energy fluorophore (Fam) is excited at 488 nm, instead of the expected fluorescence emission at 520 nm the captured energy is transferred to the lower energy fluorophore (Tamra) and is emitted at 580 nm (FRET has occurred). Using the fluorescein/rhodamine reporter/quencher combination, FRET will effectively occur even when the groups are separated by 25-30 bases of DNA. During the course of a TaqManTM assay the two fluorophores are physically detached from each other by the 5'-nuclease action of Taq DNA polymerase - after which 488 nm stimulation results in visible Fam emission at 520 nm. In this example, energy is transferred to a second heterocyclic ring which itself is a fluorophore.

In a different but related setting, energy from a fluorophore like Fam can be quenched by the heterocycle Dabcyl. In this case energy is not transferred but is lost to the local environment through interactions that require the reporter and quencher to be in near contact. Energy is not released as a second fluorescence emission but is simply dissipated as heat. Contact or near contact between reporter and quencher is required. Dabcyl quenching is employed in Molecular BeaconsTM, another popular fluorescence-based gene detection format (see technical bulletin on MOLECULAR BEACONS for more details).
Fluorescent Dye Labeled Oligonucleotides

Fluorescent dyes can be attached to oligonucleotides on the 3'-end, 5'end or even to internal residues. Multiple additions of the same or different fluorophore can be placed on a single oligo. A variety of dyes can be conjugated to oligos directly during oligo synthesis using dye-phosphoramidites or dye-CPG derivatives. An even greater variety of dyes can be conjugated to oligonucleotides post synthesis (for example, attaching a dye-NHS-ester to an amino modified oligo).

Dual-labeled probes usually have a 5'-reporter dye, such as Fam, Tet, or Hex and a 3'-quencher group, such as Tamra or Dabcyl. The 3'-group can be attached directly at the time of synthesis using a derivatized CPG (Tamra-CPG or Dabcyl-CPG) or can be attached post synthesis using NHS-ester chemistry (react a 3'-amino modified oligo with Tamra or Dabcyl-NHS-ester). Probes made using dye-CPG need only a single RP-HPLC while probes made using NHS-ester chemistry require two RP-HPLC purifications. The former approach have relatively high yield, but the later approach generates probes that have slightly lower background and greater sensitivity and are recommended for more demanding assays.

For Dual Labeled probes CLICK HERE! For Molecular Beacons CLICK HERE!

The commonly used dyes available for oligo labeling at IDT are listed below in Table 2. For excitation and emission spectra of the fluorophores, please CLICK HERE. For price information, please CLICK HERE. If you are interested in a dye not on this list please call our Technical Support team at 800-328-2661.

Table 2.
Different forms of common fluorescent dyes available for oligo labeling at IDT

  Dye Abmax (nm) Emmax (nm)
5 Phosphoramidite Conjugates Fluorescein 492 520
6-Fam 494 525
TET 521 536
HEX 535 556
Cy3 552 570
Cy5 643 667
Cy5.5 675 694
5 or 3 NHS-ester Conjugates TAMRA 565 580
ROX 585 605
JOE 520 548
Cy3 552 570
Cy5 643 667
Internal Conjugation of Any NHS-ester Dye - -
3 CPG Conjugates 6-FAM 494 525
TAMRA 565 580
Dabcyl Dark Quencher
Black HoleTM-1
Black HoleTM-2
Dark Quencher
Dyes Licensed from Molecular Probes, Inc.
(5' or 3')
Texas Red 583 603
Oregon GreenTM 492 517
*Bodipy Dyes - -
QSYTM-7 Dark Quencher
*Inquire for specific availability

Purification

As a rule, direct dye attachment through 5' phosphoramidite chemistry CLICK HERE (such as Fam, Tet or Hex) can achieve high efficiency, therefore special purification is not needed (desalting to remove free dye is standard). However, additional purification will significantly improve specific activity and sensitivity. If such purification is desired, RP-HPLC is recommended. Although simple hydrophobic cartridge can enrich the labeled product, RP-HPLC purification gives the best results.

Cy dyes are exception to the above discussion. The common Cy3 and Cy5 dyes usually have low coupling efficiency and thus demand additional purification beyond the standard desalting. RP-HPLC purification is required for these dyes.

Any oligo modified by conjugation using NHS-ester chemistry also requires RP-HPLC purification to obtain a high quality, high specific activity final product.
Guaranteed Yield

For dyes attached directly through synthesis, guaranteed yield for the 250-nmole-scale reaction is 15 OD260 units after RP-HPLC. For dyes attached by NHS-ester chemistry after synthesis, guaranteed yield after RP-HPLC is 2.0 OD260 units. An amino modifier needed for the reaction is added during synthesis and the price includes this addition.
Additional Fluorescent Dyes are Available

IDT is licensed to provide a full range of dyes from Molecular Probes, such as Texas Red and Bodipy labels. If you desire a dye-conjugated oligonucleotide using a fluorescent dye not listed above, contact IDT's Technical support Group at (800) 328-2661 or email to techsupport@idtdna.com for availability and pricing.
Mark Behlke, M.D., Ph.D.
Vice President, Molecular Genetics and Bioinformatics
Lingyan Huang, Ph.D.
Research Scientist, Molecular Genetics and Bioinformatics
Integrated DNA Technologies

March 2001

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