Fluorescein acceptor dyes

Mathies and coworkers have performed work on energy transfer using FAM, a fluorescein derivative, as the donor, and FAM, JOE, TAMRA, and ROX as the acceptors (Fig. 14.19) [111]. The acceptor dyes exhibit fairly distinct emission maxima at 525, 555, 580, and 605 nm, respectively, and all energy transfer fluorescent dye... 

Figure 2.2. The above illustration represents the configuration of tip and sample used in FRET/NSOM. The acceptor dye of the FRET pair is rhodamine. Rhodamine was incorporated into a DPPC monolayer at 0.5 mol % and used to coat an NSOM probe lacking any metal. Fluorescein was used as the donor dye in the sample, and was incorporated into two DPPC/0.5 mol % fluorescein layers separated by a spacer of three arachidic acid layers. Light exiting the tip, (blue arrow) excites the donor dye in the sample but does not directly excite the acceptor dye on the tip. When the modified NSOM probe is near the sample, however, energy transfer from the excited donor in the monolayer to the rhodamine acceptor (dark green arrow) on the tip leads to a new emission, shifted to the red (red arrow) of the donor emission (light green arrows). By monitoring rhodamine fluorescence, it is possible to optically probe only those structures within nanometers of the NSOM tip. Reproduced with permission from (Vickery et al. 1999). Copyright 1999 Biophysical Society.

A wide variety of different classes of fluorescent molecules has been investigated in the peroxyoxalate chemiluminescent systems. Among those screened were fluorescent dyes such as rhodamines and fluoresceins, heterocyclic compounds such as benzoxazoles and benzothiazoles, and a number of polycyclic aromatic hydrocarbons such as anthracenes, tetracenes, and perylenes. The polycyclic aromatic hydrocarbons and some of their amino derivatives appear to be the best acceptors as they combine high fluorescence efficiency with high excitation efficiency in the chemiluminescent reaction [28],... 

Since the same dye molecules can serve as both donors and acceptors and the transfer efficiency depends on the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, this efficiency also depends on the Stokes shift [53]. Involvement of these effects depends strongly on the properties of the dye. Fluoresceins and rhodamines exhibit high homo-FRET efficiency and self-quenching pyrene and perylene derivatives, high homo-FRET but little self-quenching and luminescent metal complexes may not exhibit homo-FRET at all because of their very strong Stokes shifts. 

Johansen et al. compared fluorescein, Eosin, Rose Bengal, and Rhodamine B. The system included the electron acceptor, methyl viologen, mv2 + which does not oxidize the dyes (nor are there dye-mv2+ complexes involved) but which reacts with the semireduced radicals formed by reduction of the dyes. The reaction scheme, in the presence of a platinum catalyst, is shown in Eqs. (32)—(35). 

A range useful of assays can be rapidly carried out using fluorescent labeled DNA. DNA can be synthesized with its 3 and 5 ends tagged with fluorescent dyes, at the 5 end 6-carboxy fluorescein (FAM) the donor, and tetramethyl-6-carboxyrhodamine (TAM) at the 3 end as the acceptor. When the two dyes are in close proximity the fluorescence is quenched, but upon separation the fluorescence is enhanced. Simple melting experiments can be designed to utilize... 

Clegg and co-workers used fluorescein as donor and tetramethylrhoda-mine as acceptor (Rq = 45 A), attaching the dyes to the 5 ends of complementary DNA strands. Others have also used fluorescein-eosin. The ability to measure the relatively long distances of a 20-mer (end-to-end distance of approximately 72 A plus linker lengths) required careful measurement and analysis of the sensitized emission (discussed above in the section on measurement). They took care to ensure that the local environment around the dyes was constant for all samples. They also adjusted the dye linker length to ensure that at least one of the dyes was rotationally mobile. (The fluorescein had a low steady-state anisotropy of... 

We have developed an energy transfer system which overcomes these difficulties. We use a luminescent lanthanide chelate as donor and an organic dye such as fluorescein, rhodamine, or CY-5 as acceptor. A number of workers have noted that the luminescent lanthanide elements terbium and europium are attractive donors because they have multiple transition dipole moments such that they act as randomized donors even in the absence of any rotational motion. This limits (1/3 < < 4/3)... 

In principle, any couple of fluorophores can be used for FRET, provided that the emission spectrum of the donor overlaps with the absorption of the acceptor. For a review of FRET-couples (and RO values) of chemical dyes see [62]. Furthermore, donors with a high fluorescence quantum-yield and acceptors with a high molar absorbance will display increased FRET. For FLIM it will be important to tune the instrument-performance to ensure maximal sensitivity to small changes in lifetimes at the control donor lifetime. Usually this is easily achieved. Many FRET-pairs have been used for FRET-FLIM including chemical probes as Fluorescein-Rhodamine [54,93],calcein-sulforhodamine B [94], and Cy3-Cy5, [70]. Since 1996, the availability of genetic-encoded fluorophores such as CFP, GFP, YFP has boosted application of FRET-FLIM enormously [95]. Nowadays fluorescent-tagging of proteins no longer depends on laborious protein pu-... 

TEOS nanoparticles doped with three types of dye were prepared for fluorescence-resonance energy transfer (FRET) by means of imposition [55]. The dyes fluorescein, 5-carboxyrhodamine 6G (R6G) and 6-carboxy-X-rhodamine (ROX) were used to prepare these TEOS nanoparticles because their spectral features effectively overlap. In the triple-dye-doped TEOS nanoparticles, fluorescein was used as a common donor for R6G and ROX, whereas R6G acted both as an acceptor for fluorescein isothiocyanate (FITG) and as a donor for ROX. To prepare the nanoparticles, the three types of amine-reactive dye molecule were first covalently linked to APS, 5-(and-6)-carboxyfluorescein succinimidyl ester, 5-carboxyrhodamine 6G succinimidyl ester and 6-carboxy-X-rhodamine succinimidyl ester. 

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