Fluorescein, fluorophore

A novel method for improving the conductivity of DNA involves close association or even incorporation of divalent metal ions such as Zn, Ni, and Co into the hehx at pH>8.5 to form metallated DNA (M-DNA). Other divalent metals such as Mg + and Ca " do not form M-DNA. In photophysical studies using ds-DNA where one strand is labeled with a fluorescein fluorophore and the complementary strand carries a rhodamine quencher, the fluorescein fluorescence is quenched only under conditions where M-DNA forms. M-DNA is an efficient conductor of electrons over distances as long as 500 basepairs and possibly as long as several micrometers. It was proposed that in M-DNA the Zn cations replace the imino protons of every thymine and guanine in addition to saturating the backbone phosphates as do other cations such as Mg (Scheme 6). Importantly, M-DNA... 

Jablonski (48-49) developed a theory in 1935 in which he presented the now standard Jablonski diagram" of singlet and triplet state energy levels that is used to explain excitation and emission processes in luminescence. He also related the fluorescence lifetimes of the perpendicular and parallel polarization components of emission to the fluorophore emission lifetime and rate of rotation. In the same year, Szymanowski (50) measured apparent lifetimes for the perpendicular and parallel polarization components of fluorescein in viscous solutions with a phase fluorometer. It was shown later by Spencer and Weber (51) that phase shift methods do not give correct values for polarized lifetimes because the theory does not include the dependence on modulation frequency. 

The nondestructive introduction of a fluorescent label would provide the molecule with a nonradioactive fluorophore, yet would preserve the option for direct radiolabelling of the fluorescent moiety with 125Iodine. This approach was pioneered by Nagasawa et al. (5) who reacted native or /V-desulfated heparins with a fluorescein isothiocyanate (FITC). The resulting degree of labelling was low... 

Kamoto and collaborators [150] designed and synthesized a new tum-on fluorescent probe 54, which is excitable by visible light (kex 490 nm), based on a fluorescein derivative 55. It is composed of a metal-nitrilotriacetic (NTA) complex as the hexahistidine tag recognition site, fluorescein as the fluorophore, and a linker. 

Fig. 4.10. (A) Schematic of percentage weights of glycerol in composite solvents corresponding to array of fluorescein solutions of varying viscosity (B) fluorescence lifetime (C) rotational correlation time images of this array and (D) plot of the rotational correlation time as a function of viscosity for this sample array exited at 470 nm the straight line fit yields a fluorophore radius of 0.54 nm for fluorescein. Adapted from Fig. 2 of Ref. [64].

Dyes which are half fluorescein, half rhodamine are called rhodols. Their spectral properties are intermediate with respect to excitation and emission wavelength. Generally, rhodol fluorophores are more... 

A fluorophore can undergo a change in their spectral properties as a result of pH variations or enzymatic activity. For example, fluorescein is such as fluorophore due to its two possible isoforms, lactone, and quinoid form. While the lactone form only absorbs in the UV and is not fluorescent, the quinoid form is excited at 490 nm and fluoresces. Only in this last form, there is... 

Another important group of hydrolytic enzymes are phospho- and cyclophosphodiesterases. They catalyze the hydrolysis of phospho-diester bonds and many of the most relevant biological substrates are nucleic acids. Phospholipase C and D are also important examples. Initial attempts to measure phosphodiesterase activity placed a phosphodiester between a fluorophore and a quencher and the probe was tested in vitro [146], This system was slightly modified by Caturla and used for the identification of catalysts with phosphodiesterase activity [147], More recently, Nagano and co-workers used a coumarin donor and fluorescein as a FRET... 

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).

The first sensor proposed for detecting gastric and oesophageal pH24, made use of two fluorophores, fluorescein and eosin, immobilised onto fibrous particles of amino-ethyl cellulose, fixed on polyester foils. Only tested in vitro, the sensor reveals a satisfactory response time of around 20 seconds. 

The intense Texas Red fluorophore has a QY that is inherently higher than the tetrameth-ylrhodamine or Lissamine rhodamine B derivatives. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes Texas Red derivatives among the best choices of labels for use in double-staining techniques. 

Aminomethylcoumarin derivatives possess intense fluorescent properties within the blue region of the visible spectrum. Their emission range is sufficiently removed from other common fluorophores that they are excellent choices for double-labeling techniques. In fact, coumarin fluorescent probes are very good donors for excited-state energy transfer to fluoresceins. 

The spectral characteristics of Lucifer Yellow iodoacetamide produce luminescence at somewhat higher wavelengths than the green luminescence of fluorescein, thus the yellow designation in its name. The excitation maximum for the probe occurs at 426 nm and its emission at 530 nm. The rather large Stoke s shift makes sensitive measurements of emission intensity possible without interference by scattered excitation light. The 2-mercaptoethanol derivative of the fluorophore has an extinction coefficient at pH 7 of about 13,000 M cm-1 at 426nm. 

In choosing a fluorescent tag, the most important factors to consider are good adsorption (high extinction coefficient), stable excitation without photobleaching, and efficient, high quantum yield of fluorescence. Some fluorophores, such as fluorescein, exhibit rapid fluorescent quenching which lowers the quantum yield over time. Up to 50 percent of the fluorescent intensity observed on a fluorescein-stained slide can be lost within 1 month in storage. AMCA and... 

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