Fluorescein and rhodamine

Fig. 6.21. Principle of detection of lipopolysaccharide (LPS) with the CD14-derived probe. It relies on the formation of a ground state complex between fluorescein and rhodamine in aqueous solution with quenching of donor and acceptor fluorescence. Spectrum A shows hypothetical fluorescence emission spectra of this complex. After LPS binding, the peptide sequence gets straightened prohibiting the close contact between the two fluorophores and leading to the recovery of red fluorescence (Spectra B).

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

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. 

McNamara et al. [38] developed water-dispersible microbeads for ratiometric imaging of pH. The 1.6 pm polystyrene beads were covered with a phospholipid layer containing covalently coupled fluorescein and rhodamine. The material was suitable for sensing pH in bulk solution and in macrophages. 

Quantum clusters are highly photostable when compared with organic fluorophores. A study was conducted to check the photostability of clusters in comparison to organic fluorophores and semiconductor quantum dots [12]. Photostability of a gold cluster capped with dihydrolipoic acid (AuNC DHLA) was compared with polymer coated CdSe/ZnS semiconductor quantum dots and two different organic fluorophores namely fluorescein and rhodamine 6G (Fig. 5). For the study, 20 pi of fluorescent AuNC DHLA was dissolved in sodium borate buffer of pH 9. The sample was loaded into a quartz cuvette and was exposed to blue-light (480 nm)... 

Triplet state kinetics can also be studied by FCS (Widengren et al., 1995). In fact, with dyes such as fluoresceins and rhodamines, additional fluctuations in fluorescence are observed when increasing excitation intensities as the molecules enter and leave their triplet states. The time-dependent part of the autocorrelation function is given by... 

For practical applications it is better to use fluorescent probes excitable at high wavelengths, so that autofluorescence of the sample (especially biological samples) does not interfere. Calcium indicators based on fluorescein and rhodamine fluorophores(70) fulfill this requirement and are thus more convenient for fluorescence... 

In the late 1960s, Gerischer and Tributsch researched a ZnO photoelectrode sensitized by organic dyes, including rose bengal, fluorescein, and rhodamine ... 

The N-terminal amino group or a Lys residue can easily be used to label the PNA. This can be carried out in solution after purification, or more conveniently, while the PNA is still attached to the resin. Fluorescein and rhodamine are the most common fluorophore labels, while biotin is the most common affinity tag. Fluorescein or rhodamine are usually coupled to the amino group as -OSu esters or isothiocyanates. 4,4 -Dimethoxytrityl-protected biotin and Piv-pro-tected fluorescein have also been coupled to the N-terminus of a PNA as their 1-phenylpyr-azolin-5-one carboxylate esters.147 In 1997 a new fluorescein-conjugated Lys monomer (21, Scheme 12) was described. 48 ... 

Antibodies labeled with fluorescent molecules have several applications, particularly in cytochemistry and cell sorting. There are many fluorochromes used in labeling (1), such as coumarin derivatives, phycobiliproteins, and rare earth chelates however, fluorescein and rhodamine (Table 1) are the most commonly used. 

The isothiocyanate derivatives of fluorescein and rhodamine are widely used to label antibodies. They react with the amino groups of the immunoglobin G (IgG) molecule under alkaline conditions and a molar excess of about 20 is usually optimal. More recently, N-... 

FIGURE 3.38 A micromixer based on distributive mixing. Flow visualization using fluorescein and rhodamine B at a total flow rate of 50 mL/min. Only 10 out of 16 ministreams are shown [466]. Reprinted with permission from the Royal Society of Chemistry. 

These bind covalently to lysines and cysteines of proteins, and absorb and fluoresce in the visible. The fluorescence lifetimes of fluorescein and rhodamine are around 4 ns, and their emission spectra are not sensitive to medium polarity. 

R. Tschesche, Fortschr. Chem. Org. Naturstoffe 4, 1-27 (1945). Fluoran, fluoresceins, and rhodamines ... 

The model of ionic binding was also evaluated by comparing the binding of fluorescein and rhodamine to hair, two molecules of similar shape, size, and fluorescent quantum yield. Rhodamine, being positively charged, is far more efficient at binding to hair compared to fluorescein. The disparity in binding of materials to... 

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