Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5

The amine-reactive fluorescein derivatives (Amine-reactive xanthene derivatives in this section - Table 1.8) have been the most common fluorescent derivatization reagents for covalently labeling proteins. In addition to its relatively high absorptivity, excellent fluorescence quantum yield and good water solubility, fluorescein (F1300, ) has an excitation maximum (494 nm) that closely matches the 488 nm spectral line of the argon-ion laser, making it an important fluorophore for confocal laser-scanning microscopy and flow cytometry applications. In addition, fluorescein's protein conjugates are not inordinately susceptible to precipitation. Because it can be prepared in high purity, fluorescein is one of the five dyes in our Reference Dye Sampler Kit (R14782, Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1). Molecular Probes is also the source of the NIST-traceable fluorescein standard (F36915) described below.

Unfortunately, fluorescein-based dyes and their conjugates have several drawbacks, including:

• A relatively high rate of photobleaching (Figure 1.9, , , Figure 1.46, Figure 1.53, Figure 7.23, Figure 11.8)
• pH-sensitive fluorescence (pK_a ~6.4) that is significantly reduced below pH 7 (Figure 1.12)
• A relatively broad fluorescence emission spectrum (Figure 1.43), limiting their utility in some multicolor applications
• A tendency toward quenching of their fluorescence on conjugation to biopolymers, particularly at high degrees of substitution (Figure 1.54)

The photobleaching and pH sensitivity of fluorescein makes quantitative measurements with this fluorophore problematic. Furthermore, fluorescein's relatively high photobleaching rate limits the sensitivity that can be obtained, a significant disadvantage for applications requiring ultrasensitive detection, such as DNA sequencing (Labeling Oligonucleotides and Nucleic Acids - Section 8.2), fluorescence in situ hybridization (Detecting Nucleic Acid Hybridization - Section 8.5) and localization of low-abundance receptors. These limitations have encouraged the development of alternative fluorophores. However, because of the widespread availability of optical filter sets designed to efficiently excite and detect fluorescein's fluorescence (Optical Filters for Fluorescence Microscopy - Section 23.5, Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11) and the near-optimal match of fluorescein dyes to the 488 nm spectral line of the argon-ion laser, useful fluorescein substitutes must closely replicate fluorescein's spectra.

There are no new dyes available that completely solve fluorescein's photobleaching problems, but Molecular Probes has developed some excellent dyes whose spectra mimic those of fluorescein — the Alexa Fluor 488 (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum - Section 1.3), BODIPY FL (BODIPY Dye Series - Section 1.4), Oregon Green 488, Oregon Green 514 and Rhodamine Green dyes (this section). These dyes are much more photostable than fluorescein and have less or no pH sensitivity in the physiological pH range. When compared with fluorescein, all of these dyes exhibit the same or slightly longer-wavelength spectra (absorption maxima ~490–515 nm) and comparably high fluorescence quantum yields. Alternatively, where they can be used, our yellow-green fluorescent FluoSpheres microspheres (Microspheres - Section 6.5) provide a means of preparing bioconjugates that have a combination of fluorescence intensity and photostability far superior to that of any simple dye conjugate.

The National Institute of Standards and Technology (NIST) chose a high-grade fluorescein synthesized by Molecular Probes to create Standard Reference Material 1932 (SRM 1932), a certified fluorescein solution. Molecular Probes now offers a NIST-traceable fluorescein standard (F36915) that not only meets the stringent criteria established by NIST, but is also directly traceable to SRM 1932. We supply our NIST-traceable fluorescein standard as a calibrated 50 µM solution of fluorescein in 100 mM sodium borate buffer, pH 9.5; under these conditions, fluorescein is completely ionized and is therefore in its most fluorescent form (Figure 20.1, Figure 20.2), exhibiting an extremely high quantum yield of 0.93 (Probes Useful at Near-Neutral pH - Section 20.2).

Figure 20.1 Ionization equilibria of fluorescein.

Figure 20.2 The pH-dependent spectra of fluorescein (F1300): A) absorption spectra, B) emission spectra.

Academic researchers and industry scientists alike can use our NIST-traceable fluorescein standard to assess day-to-day or experiment-to-experiment variation in fluorescence-based instrumentation, as well as to determine the Molecules of Equivalent Soluble Fluorophore (MESF) value for an experimental solution. The MESF value is defined not as the actual number of dye molecules present, but rather as the number of fluorophores that would yield a fluorescence intensity equivalent to that of the experimental solution when analyzed on the same instrument under the same conditions. Consequently, the MESF value is an important tool for characterizing the fluorescence intensity of a solution containing spectrally similar dye molecules attached to antibodies, nucleic acids, microspheres or other substrates that might enhance or diminish the fluorescence. When its pH is carefully matched with that of the experimental solution, our NIST-traceable fluorescein standard can be used for accurate MESF determinations of a wide range of green-fluorescent dye solutions and on an assortment of fluorescence-based instruments.

Single-Isomer Fluorescein Isothiocyanate (FITC) Preparations

Despite the availability of alternative amine-reactive fluorescein derivatives that yield conjugates with superior stability and comparable spectra, fluorescein isothiocyanate (FITC) remains one of the most popular fluorescent labeling reagents. The synthesis of fluorescein isothiocyanate, carboxyfluorescein (FAM, see below) and similar fluorescein-derived reagents yields a mixture of isomers at the 5- and 6-positions of fluorescein's "bottom" ring (). Spectra of the two isomers are almost indistinguishable in both wavelength and intensity. However, the isomers may differ in the geometry of their binding to proteins, and the conjugates may elute under different chromatographic conditions or migrate differently in an electrophoretic gel when the dyes are used for high-resolution DNA sequencing. Thus, certain applications may require the single-isomer preparations. Many fluorescein (and rhodamine) probes are available from Molecular Probes either as a mixture of isomers or as purified single isomers.

The 5-isomer or "isomer I" of FITC (F143, , ) is the most widely used FITC isomer, probably because it is easier to isolate in pure form. Because isothiocyanates may deteriorate during storage, we recommend purchasing the 5-isomer of FITC specially packaged in individual vials (F1906, F1907). FITC is readily soluble in aqueous solutions that have a pH above 6. FITC is also available in our FluoReporter FITC Protein Labeling Kit (F6434, Active esters and kits for labeling proteins and nucleic acids - Table 1.2, FluoReporter(R) FITC Protein Labeling Kit). This kit and its components are described in Kits for Labeling Proteins and Nucleic Acids - Section 1.2.

In addition to its widespread use for preparing immunoreagents, FITC has a multitude of other applications. Oligonucleotide conjugates of FITC are frequently employed as hybridization probes. Peptide conjugates of FITC and other fluorescent isothiocyanates are susceptible to Edman degradation, making them useful for high-sensitivity amino acid sequencing; FITC-labeled amino acids and peptides have been separated by capillary electrophoresis, with a detection limit of fewer than 1000 molecules. FITC has also been used to detect proteins in gels and on nitrocellulose membranes, and FITC is a selective inhibitor of several membrane ATPases. Furthermore, fluorescein-to-fluorescein excited-state energy transfer leads to self-quenching (Fluorescence Resonance Energy Transfer (FRET) - Note 1.2 ). This self-quenching has permitted scientists to follow the assembly of fluorescein-labeled C9 complement protein from its subunits. The degree of substitution of proteins by FITC has been accurately determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. FITC — and probably Oregon Green isothiocyanate (O6080) and eosin isothiocyanate (E118, see below) — at a concentration of 2–500 nM can be used as a highly selective marker of eosinophils.

Mixed-Isomer and Single-Isomer Preparations of Carboxyfluorescein (FAM) Succinimidyl Ester

Although many other companies still prepare their fluorescein bioconjugates with FITC, Molecular Probes prefers to use amine-reactive succinimidyl esters of carboxyfluorescein (commonly called FAM), which yield carboxamides that are more resistant to hydrolysis. We offer both mixed-isomer and single-isomer preparations of FAM (FluoroPure Grade - Note 19.2, C1904; C1359, C1360) and FAM succinimidyl esters (C1311, C2210, C6164). A study comparing the relative conjugation rate of several reactive fluorescein derivatives with a protein or L-lysine and the stability of the resulting conjugates concluded that the succinimidyl ester of carboxyfluorescein showed superior performance, followed by fluorescein dichlorotriazine (DTAF, see below). FITC was both the slowest to react and yielded the least stable conjugates; however, the degree of labeling was most easily controlled with FITC. The succinimidyl ester of 5-FAM (C2210) is reported to react much faster than FITC when used to derivatize small biomolecules prior to separation by capillary electrophoresis.

Succinimidyl Esters of Fluorescein with Spacer Groups

We also prepare succinimidyl esters of fluorescein that contain aliphatic spacers between the fluorophore and the reactive group. These include mixed-isomer (F2181, F6129) and single-isomer (F6106) preparations of fluorescein-X succinimidyl ester (SFX), which contains a seven-atom aminohexanoyl spacer ("X") between the FAM fluorophore and the succinimidyl ester (). In addition, we offer fluorescein-5-EX succinimidyl ester (F6130), which contains a seven-atom spacer that is somewhat more hydrophilic than is the spacer in SFX (). These spacers separate the fluorophore from the biomolecule to which it is conjugated, potentially reducing the quenching that typically occurs upon conjugation. We have determined that conjugates of some proteins prepared with fluorescein-5-EX succinimidyl ester are up to twice as fluorescent as the corresponding conjugates labeled with FITC at the same degree of labeling (Figure 1.54). Consequently, we now recommend this fluorescein derivative as the preferred dye for preparing most fluoresceinated proteins. Fluorescein-5-EX succinimidyl ester is also available in our convenient FluoReporter Fluorescein-EX Protein Labeling Kit (F6433) and Fluorescein-EX Protein Labeling Kit (F10240). See Kits for Labeling Proteins and Nucleic Acids - Section 1.2 and Molecular Probes' kits for protein and nucleic acid labeling - Table 1.3 for more details about these labeling kits.

The spacers in our SFX and fluorescein-5-EX succinimidyl esters may also make the fluorophore more accessible to secondary detection reagents. For example, the spacers should make the fluorescein moiety more available for quenching by our polyclonal and monoclonal anti-fluorescein/Oregon Green antibodies, a technique used to determine the accessibility of the fluorophore in proteins, membranes and cells. Fluorescein is frequently used as a hapten on a primary detection reagent that can be either amplified or converted into a longer-wavelength or electron-dense signal with the appropriate secondary detection reagent. Anti-Dye and Anti-Hapten Antibodies - Section 7.4 describes our extensive selection of antibodies to fluorescein and other dyes.

Fluorescein Dichlorotriazine (DTAF)

The 5-isomer of fluorescein dichlorotriazine (5-DTAF, D16) is highly reactive with proteins and is commonly used to prepare biologically active fluorescein tubulin. Unlike other reactive fluoresceins, 5-DTAF also reacts directly with polysaccharides and other alcohols in aqueous solution at pH above 9, but cannot be used to modify alcohols in the presence of better nucleophiles such as amines or thiols. Polysaccharides that have been modified by DTAF (or other fluorescein derivatives) are readily radioiodinated.

Caged Fluorescein

"Caged" probes are those that can liberate an active species upon illumination with ultraviolet light (Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes - Section 5.3). Caged versions of nucleotides, drugs and ion indicators are particularly common. Caged fluorescent dyes can be utilized as polar tracers whose fluorescence can be spatially and temporally "turned on" by illumination. Conjugation of the succinimidyl ester of our water-soluble, caged carboxyfluorescein β-alanine-carboxamide (C20050, ) to a biomolecule of interest produces an essentially nonfluorescent probe that yields a green-fluorescent fluorescein-labeled product only after ultraviolet illumination. We have utilized this amine-reactive reagent to prepare conjugates of goat anti–mouse IgG and goat anti–rabbit IgG antibodies (G21061, G21080; Secondary Immunoreagents - Section 7.2). Unlike dye-labeled antibodies, brief ultraviolet illumination of these conjugates results in an increase in fluorescence at the labeling site, a property that may be useful in overcoming high autofluorescence in the sample. Furthermore, photolysis of caged fluorescein conjugates releases a fluorescein dye that can serve as a hapten for our anti-fluorescein/Oregon Green antibodies (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Figure 7.71).

Figure 7.71 Schematic representation of photoactivated fluorescence combined with sample masking. Initially, no fluorescence is observed from samples stained with a CMNB-caged fluorescein-labeled secondary detection reagent (panel A). The desired mask is then placed over the sample (panel B), after which the sample is exposed to UV light. The mask is then removed; fluorescein molecules present in the unmasked portion of the sample are uncaged by the UV light and fluoresce brightly when viewed with the appropriate filters (panel C). Uncaged fluorescein may now also serve as a hapten for further signal amplification using our anti-fluorescein/Oregon Green antibody (A889). For example, probing with the anti-fluorescein/Oregon Green antibody followed by staining with the Alexa Fluor 594 goat anti–mouse IgG antibody (A11005) can be used to change the color of the uncaged probe to red fluorescent (panel D).

Spectral Properties of the Oregon Green Dyes

Our Patented Oregon Green 488 and Oregon Green 514 dyes are fluorinated analogs of fluoresceins. The absorption and emission spectra of the Oregon Green 488 dye (2',7'-difluorofluorescein; D6145) perfectly match those of fluorescein (). With additional fluorination of the "bottom" ring of fluorescein, the Oregon Green 514 dye exhibits a moderate shift in its absorption and fluorescence spectra of about 15 nm relative to those of fluorescein or the Oregon Green 488 dye. Because of the near match of their absorption maxima on proteins (~498 nm and ~512 nm) to the strong 488 nm and 514 nm spectral lines of the argon-ion laser, the Oregon Green 488 and Oregon Green 514 fluorophores are important dyes for both confocal laser-scanning microscopy and flow cytometry applications. Furthermore, sophisticated detection systems, such as the Zeiss META system, use linear-unmixing software to differentiate between fluorescence emission maxima <5 nm apart, greatly expanding the palette of fluorescent colors available for multicolor labeling experiments and permitting the use of the Oregon Green 514 dye in combination with other green-fluorescent dyes.

Advantages of the Oregon Green Dyes

Bioconjugates prepared from the Oregon Green 488 and Oregon Green 514 dyes share several advantages over those of other fluorescein dyes. These include:

• Fluorescence of protein conjugates prepared from the Oregon Green 488 and Oregon Green 514 dyes is not appreciably quenched, even at relatively high degrees of labeling (Figure 1.54).
• Conjugates of the Oregon Green 488 and Oregon Green 514 fluorophores are more photostable than those of fluorescein (Figure 1.46). The superior photostability of the Oregon Green 488 dye and, in particular, the Oregon Green 514 conjugates permits the acquisition of many more photons before the photodestruction of the dye, making the Oregon Green dyes particularly useful substitutes for fluoresceins for fluorescence imaging applications ().
• Oregon Green dyes have a lower pK_a (pK_a = 4.7 versus 6.4 for fluorescein) (Figure 1.12), making their fluorescence essentially pH insensitive in the physiological pH range. However, the pH sensitivity of the Oregon Green dyes in the weakly acidic range (pH 4 to 6) also makes these dyes useful as pH indicators for acidic organelles of live cells (Probes Useful at Acidic pH - Section 20.3).
• Oregon Green dyes are excellent haptens for anti-fluorescein/Oregon Green antibodies (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Selected haptenylation reagents and their anti-hapten antibodies - Table 4.2), making Oregon Green bioconjugates useful in a variety of signal amplification schemes.

Both Oregon Green 488 and Oregon Green 514 dyes have also proven useful as fluorescence anisotropy probes for measuring protein–protein and protein–nucleic acid interactions.

Reactive Oregon Green Dyes

We have prepared a variety of reactive derivatives that enable researchers to take advantage of the excellent spectral properties of the Oregon Green 488 and Oregon Green 514 dyes (Amine-reactive xanthene derivatives in this section - Table 1.8). These include the FITC analog, Oregon Green 488 isothiocyanate (F₂FITC, O6080), and the single-isomer succinimidyl esters of Oregon Green 488 carboxylic acid (O6147, O6149) and Oregon Green 514 carboxylic acid (O6139), as well as the 5-isomer of Oregon Green 488 carboxylic acid (O6146, ) and the mixed-isomer preparation of Oregon Green 514 carboxylic acid (O6138, ). The 6-isomer of Oregon Green 488-X succinimidyl ester (O6185, ) contains a seven-atom aminohexanoyl spacer ("X") between the fluorophore and the succinimidyl ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated and making it more accessible to secondary detection reagents, such as anti-dye antibodies (Anti-Dye and Anti-Hapten Antibodies - Section 7.4). Oregon Green 488 iodoacetamide (O6010) and Oregon Green 488 maleimide (O6034), which are useful for thiol conjugation, are described in Thiol-Reactive Probes Excited with Visible Light - Section 2.2. We also offer Oregon Green 488 cadaverine (O10465, Derivatization Reagents for Carboxylic Acids and Glutamine - Section 3.3) for synthesizing conjugates and labeling carboxylic acids.

The Oregon Green fluorophores, reactive dyes and conjugates are Patented by Molecular Probes, Inc., and are offered for research purposes only. Molecular Probes welcomes inquiries about Licensing these products for resale or other commercial uses. Custom conjugations of the Oregon Green 488 fluorophore are also available. Please contact our Custom and Bulk Sales Department.

To facilitate direct labeling of biomolecules, we offer several types of labeling kits that incorporate reactive versions of our Oregon Green dyes. These kits are easy to use and give reliable conjugations in minimal time. Our Oregon Green protein and nucleic acid labeling kits, which are described in detail in the indicated sections, include the:

• FluoReporter Oregon Green 488 and Oregon Green 514 Protein Labeling Kits (F6153, F6155; Kits for Labeling Proteins and Nucleic Acids - Section 1.2; FluoReporter(R) Oregon Green(R) 488 Protein Labeling Kit, FluoReporter(R) Oregon Green(R) 514 Protein Labeling Kit)
• Easy-to-Use Oregon Green 488 Protein Labeling Kit (O10241, Kits for Labeling Proteins and Nucleic Acids - Section 1.2, Oregon Green(R) 488 Protein Labeling Kit)
• Zenon Oregon Green 488 Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14)
• ULYSIS Oregon Green 488 Nucleic Acid Labeling Kit (U21659, Labeling Oligonucleotides and Nucleic Acids - Section 8.2, ULYSIS Nucleic Acid Labeling Kits)

Oregon Green 488 Tyramide Signal Amplification Kits

Tyramide signal amplification (TSA) utilizes horseradish peroxidase conjugates to yield significant amplification of targets (Figure 6.5). Our TSA Kits #9 (T20919) and #29 (T20939), which are described in Tyramide Signal Amplification (TSA) Technology - Section 6.2, contain Oregon Green 488 tyramide and horseradish peroxidase conjugates of either goat anti–mouse IgG antibody or streptavidin. Once deposited, the Oregon Green 488 tyramide can serve as a hapten for further amplification by using a second round of TSA (Figure 6.5) or our ELF technology (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3).

When directly compared with their fluorescein analogs, Oregon Green 488 and Oregon Green 514 conjugates typically have higher fluorescence yields and greater resistance to photobleaching. We have used succinimidyl esters of the Oregon Green 488 and Oregon Green 514 carboxylic acids to prepare conjugates of:

• Antibodies (Summary of Molecular Probes' secondary antibody conjugates - Table 7.1) and protein A (Protein A and protein G conjugates - Table 7.13), which are described in Secondary Immunoreagents - Section 7.2
• Streptavidin and NeutrAvidin biotin-binding protein (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23)
• Lectins (Lectins and Other Carbohydrate-Binding Proteins - Section 7.7, Molecular Probes' selection of lectin conjugates - Table 7.24)
• ChromaTide dUTP (C7630, Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Characteristics of ChromaTide dUTP, ChromaTide OBEA-dCTP, aha-dUTP and aha-dCTP labeled nucleotides - Table 8.7) for synthesis of labeled DNA
• Phalloidin and DNase I (Probes for Actin - Section 11.1, Spectral characteristics of our F-actin-selective probes - Table 11.1, ) for staining actin in fixed cells
• Tubulin (T12391, Probes for Tubulin and Other Cytoskeletal Proteins - Section 11.2)
• Paclitaxel (Taxol) for staining tubulin filaments in live cells (Flutax-2, P22310; Probes for Tubulin and Other Cytoskeletal Proteins - Section 11.2; )
• DHPE, a phospholipid (O12650, Fatty Acid Analogs and Phospholipids - Section 13.2)
• Biocytin (O12920, Polar Tracers - Section 14.3)
• Dextrans (Fluorescent and Biotinylated Dextrans - Section 14.5, Molecular Probes' selection of dextran conjugates - Table 14.4)
• Annexin V (A13200, Assays for Apoptosis - Section 15.5)
• Polymyxin B (P13236, Viability and Cytotoxicity Assay Reagents - Section 15.2)
• Collagen IV and gelatin (C13185, G13186; Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione - Section 15.6)
• Transferrin (T13341), epidermal growth factor (E7498) and fibrinogen (F7496). See Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1 for details on these products.
• α-Bungarotoxin (B7488, Probes for Neurotransmitter Receptors - Section 16.2)
• Shuttle PIP carriers for transport of fluorescent phosphatidylinositol polyphosphates into living cells (Probes for Lipid Metabolism and Signaling - Section 17.4, )
• BAPTA, a calcium chelator (Fluorescent Ca{2+} Indicators Excited with Visible Light - Section 19.3, Fluorescent Ca{2+} Indicator Conjugates - Section 19.4)

In addition to the single isomers of the succinimidyl ester of carboxyfluorescein, 5-FAM (C2210) and 6-FAM (C6164), Molecular Probes offers the fluorescein derivatives JOE, HEX and TET for genetic analysis (Figure 1.65). These dyes are important for automated DNA sequencing applications. They are also commonly used as fluorescent donors to label primers and hybridization probes (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Detecting Nucleic Acid Hybridization - Section 8.5; Amine-reactive dyes for nucleic acid sequencing - Table 8.11), often in combination with the rhodamine-based fluorescent acceptors ROX (C6125, C6126) and TAMRA (C6121, C6122; Amine-reactive dyes for nucleic acid sequencing - Table 8.11). The nonfluorescent quenchers dabcyl (D2245), dabsyl (D1537) and the QSY dyes (Molecular Probes' nonfluorescent quenchers and photosensitizers - Table 1.10) can also be used as energy acceptors in conjunction with these fluorophores. Furthermore, sophisticated detection systems, such as the Zeiss META system, use linear-unmixing software to differentiate between fluorescence emission maxima <5 nm apart, greatly expanding the palette of fluorescent colors available for multicolor labeling experiments and permitting the use of these dyes and their conjugates in combination with other green- or orange-fluorescent dyes.

Figure 1.65 Normalized emission spectra of 5-FAM SE (C2210, green), 6-TET SE (C20092, orange), 6-JOE SE (C6171MP, red), and 6-HEX SE (C20091, blue).

JOE

Chemical modifications of the xanthene ring of fluoresceins typically shift the dye's absorption and emission maxima to longer wavelengths (Figure 1.65). We offer a single-isomer preparation of the succinimidyl ester of 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (6-JOE, SE; C6171MP; Figure 1.66). 6-JOE is one of the traditional fluorophores (i.e., 5-FAM, 6-JOE, 6-TAMRA and 6-ROX) used in automated DNA sequencing (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Amine-reactive dyes for nucleic acid sequencing - Table 8.11).

Figure 1.66 Structures of 6-JOE SE (C6171MP), 6-HEX SE (C20091) and 6-TET SE (C20092).

TET

Like JOE, the succinimidyl ester of 6-carboxy-2',4,7,7'-tetrachlorofluorescein (TET, SE; C20092) has a chlorinated xanthene ring, but also additional chlorination of the "bottom" ring (Figure 1.66). As a result, TET has red-shifted absorption and emission maxima of 521 and 536 nm, respectively (Figure 1.65). TET and FAM are often used simultaneously as FRET donors to TAMRA for RT-PCR and SSP-PCR applications.

HEX

With excitation and emission maxima of 535 and 556 nm, respectively, the isomer-free succinimidyl ester of 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEX, SE; C20091) has the longest wavelengths of these chlorinated fluorescein derivatives (Figure 1.65). The HEX dye has four chlorine atoms on the xanthene ring and two on the lower ring (Figure 1.66). HEX is often employed in multiplexed DNA sequencing for classical genotyping (Labeling Oligonucleotides and Nucleic Acids - Section 8.2, Amine-reactive dyes for nucleic acid sequencing - Table 8.11) and in pathological forensics. HEX has also been used in conjunction with the FAM and TET dyes in a 5'-exonuclease assay to detect three different Candida species in a single reaction tube.

Eosin and Erythrosin

The reactive eosin (2',4',5',7'-tetrabromofluorescein) and erythrosin (2',4',5',7'-tetraiodofluorescein) dyes are usually not chosen for their fluorescence properties — the fluorescence quantum yield of eosin is typically only about 10–20% that of fluorescein, and erythrosin is even less fluorescent — but rather for their ability to act as phosphorescent probes or as photosensitizers. With their high quantum yields (~0.57) for singlet oxygen generation, eosin and its conjugates can be used as effective photooxidizers of diaminobenzidine (DAB) in high-resolution electron microscopy studies (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents - Note 14.2). Like their thiol-reactive counterparts in Thiol-Reactive Probes Excited with Visible Light - Section 2.2, eosin and erythrosin isothiocyanates (E18, E30150) are particularly useful as phosphorescent probes for measuring the rotational properties of proteins, virus particles and other biomolecules in solution and in membranes. In addition, they are employed for fluorescence resonance energy transfer (FRET) studies (Fluorescence Resonance Energy Transfer (FRET) - Note 1.2 ) and for fluorescence recovery after photobleaching (FRAP) measurements of lateral diffusion.

An Eosin Analog

In 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein, the carboxylic acid usually found in eosin dyes is replaced by a sulfonic acid (). The resulting dye is somewhat more photostable than eosin, but is likely to have a similar triplet yield. Because the ability to generate singlet oxygen is lost when a dye bleaches, it is possible that conjugates prepared from the succinimidyl ester of this dye (C6166) will produce singlet oxygen for longer periods, potentially making them more useful than eosin conjugates for photoconversion studies.

Reactive Rhodamine Green Dyes

The Rhodamine Green dye, which is the nonsulfonated analog of our important Alexa Fluor 488 dye, offers a combination of desirable properties, including good photostability, a high extinction coefficient (>75,000 cm^-1M^-1) and a high fluorescence quantum yield, particularly in its nucleotide and nucleic acid conjugates. The Rhodamine Green fluorophore — our trademark for carboxyrhodamine 110 — is even more photostable than the Oregon Green 488 dye and about equivalent in photostability to the Oregon Green 514 dye (Figure 1.46). Moreover, the fluorescence of its conjugates is completely insensitive to pH between 4 and 9 (Figure 1.12).

Reactive versions of the Rhodamine Green dye (Amine-reactive xanthene derivatives in this section - Table 1.8) were originally developed by Molecular Probes for use in DNA sequencing and other applications. Conjugates of the Rhodamine Green fluorophore with amines can be prepared either directly from its succinimidyl ester (5(6)-CR 110, SE; R6107) or indirectly from its TFA-protected derivative (5(6)-CR 110 TFA, SE; R6112; Figure 1.68). The succinimidyl ester of the Rhodamine Green-X dye (R6113) has an additional seven-atom aminohexanoyl spacer ("X") to potentially reduce interaction of the fluorophore and its reaction site. The absorption and fluorescence emission maxima of Rhodamine Green conjugates are red-shifted about 7 nm compared with those of fluorescein; however, they remain compatible with standard fluorescein optical filter sets (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11). The Rhodamine Green fluorophore has been used to label the peptide gastrin; however, in general, Rhodamine Green succinimidyl esters are much less suitable for protein conjugations than are succinimidyl esters of the Alexa Fluor and Oregon Green dyes. Rhodamine Green dye–labeled probes have been frequently used for fluorescence correlation spectroscopy (Fluorescence Correlation Spectroscopy (FCS) - Note 1.4 ).

Figure 1.68 Conjugation of the succinimidyl ester of Rhodamine Green TFA (R6112) to an amine, followed by deprotection of the fluorophore with either hydroxylamine or ammonia.

Rhodamine Green Conjugates

Although the Rhodamine Green dye is one of the most photostable of the fluorescein substitutes, its fluorescence when conjugated to proteins is often substantially quenched, and these conjugates also tend to precipitate from solution. Therefore, we do not recommend any of the Rhodamine Green succinimidyl esters for preparing protein conjugates. However, when conjugated to dextrans, nucleotides and oligonucleotides, the Rhodamine Green fluorophore remains quite fluorescent. Molecular Probes currently has available Rhodamine Green dextran conjugates (Fluorescent and Biotinylated Dextrans - Section 14.5, Molecular Probes' selection of dextran conjugates - Table 14.4) and ChromaTide Rhodamine Green dUTP (C7629; Labeling Oligonucleotides and Nucleic Acids - Section 8.2; Characteristics of ChromaTide UTP nucleotides - Table 8.6, Characteristics of ChromaTide dUTP, ChromaTide OBEA-dCTP, aha-dUTP and aha-dCTP labeled nucleotides - Table 8.7).