Fluorescein spectra

Addition of NaDS (li = 4.10 ) perturbs the fluoresceine spectrum in the direction of an extensive displacement of dye molecules from the protein with an apparent "equivalence-point corresponding to a stoichiometric N/(hsa] ratio close to 15, according to absorption and fluorescence data (see Fig. 5 ). At pH = 6.5, on the contrary, any effect was barely detectable. This is in... 

Fluorescein is excited at 494 nm, which fits to the argon-ion laser line at 488 nm, a very convenient feature for many microscopy experiments. It emits at 520 nm and the emission band is far from being sharp. The broad fluorescence emission spectrum varies with pH [18]. The advantageous photochemical properties of fluorescein are its high absorption (emax = 79,000M-1cm-1) and quantum... 

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 spectral characteristics of protein conjugates made with Lissamine rhodamine B derivatives are of longer wavelength than those of tetramethylrhodamine—more toward the red region of the spectrum. In addition, modified proteins have better chemical stability and are somewhat easier to purify than those made from TRITC (discussed previously). Lissamine derivatives also make more photostable probes than the fluorescein derivatives (Section 1, this chapter). 

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. 

DPA) in dimethylphthalate at about 70°, yields a relatively strong blue Umax =435 nm) chemiluminescence the quantum yield is about 7% that of luminol 64>. The emission spectrum matches that of DPA fluorescence so that the available excitation energy is more than 70 kcal/mole. Energy transfer was observed on other fluorescers, e.g. rubrene and fluorescein. The mechansim of the phthaloyl peroxide/fluorescer chemiluminescence reaction very probably involves radicals. Luminol also chemiluminesces when heated with phthaloyl peroxide but only in the presence of base, which suggests another mechanism. The products of phthaloyl peroxide thermolysis are carbon dioxide, benzoic acid, phthalic anhydride, o-phenyl benzoic acid and some other compounds 65>66>. It is not yet known which of them is the key intermediate which transfers its excitation energy to the fluorescer. 

The first, and still the most commonly used, of the tunable lasers were those based upon solutions of organic dyes. The first dye laser was developed by Sorokin and Lankard 05), and used a "chloro-aluminum phthalocyanine" (sic) solution. Tunable dye lasers operating throughout the visible spectrum were soon produced, using dyes such as coumarins, fluorescein, rhodamines, etc. Each dye will emit laser radiation which is continuously tunable over approximately the fluorescence wavelength range of the dye. 

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. 

Figure 8.21. Fluorescence spectra of fluorescein on an alumina-TLC plate. Upper redshift of the spectra with concentration according to increasing fluorescence reabsorption. Lower corrected fluorescence spectrum of the most concentrated sample (dashed line) according in Figure 8.20.

This procedure, which complements other methods for distinguishing apoptotic and necrotic cell death, employs annexin V-PE as a marker for early apoptotic cells, and 7-AAD for late apoptotic or necrotic cells. Although other versions of this assay have used annexin V-fluorescein together with PI, that combination precludes the use of a third fluorescence color to measure an additional parameter, such as a phenotypic marker, because PI, unlike 7-AAD, has a broad emission spectrum that includes both orange and red fluorescence. 

The quantification of fluorescent particles in cellular systems is difficult because several aspects such as autofluorescence, bleaching (see below), and quenching hamper analysis. Keep in mind that many fluorophores show a pH-dependent change in emission spectrum and intensity fluorescein-labeled dextrans (FITC-dextran) and calcein are strongly quenched upon acidification. If available, one should read the fluorescence intensity at its isosbestic point, where the intensity is not pH dependent. 

Fig, (, Excitation and emission spectra of the fluorescein derivative DTAF. Modified from ref. I. EX, excitation spectrum EM, emission spectrum. 

Rhodamine is a preferred fluorochrome over fluorescein because of its slower bleach rate and its emission in a spectrum that shows less cellular autofluorescence. Also, this spectrum produces less autofluorescence in plastic substrata (see Chapter 14). Rhodamine requires a mercury vapor light source, since other sources, such as xenon, do not have sufficient emission in the green spectrum. 

The main fluorescent pH indicator probes are based on fluorescein and therefore it is important to understand the pH-dependent ionic equilibria of it and its derivatives, hi aqueous solutions above pH 9 the phenolic and carboxylic acid functional groups in the molecule are almost totally ionised (Figure 3.14). Upon acidification of the dianion, firstly, protonation of the phenolic group occurs (pK 6.4) to yield the monoanion followed by the carboxylic acid (pA < 5), giving the neutral species of fluorescein. On further acidification the fluorescein cation pK 2.1) is generated. In strongly acidic environments fluorescein is non-fluorescent, only the mono-anion and di-anions are fluorescent, with quantum yields of 0.37 and 0.93, respectively. The pH-dependent absorption spectrum of fluorescein exhibits a blue-shift and... 

Fig. 7 Selected 2PA spectra obtained by absolute fluorescence-based methods, a Spectra for fluorescein, rhodamine B, coumarin 307, and , -p-bis(o-methylstyryl)benzene (the solvent is indicated in the legend) obtained by Xu and Webb [78]. In the case of coumarin 307, the ordinate displays the quantity rjSy where rj is the fluorescence quantum yield, b Spectrum for , -p-bis(o-methylstyryl)benzene (this spectrum is obtained from the tabulated values for the band shape reported by Kennedy and Lytle [90] and the cross section at 585 nm reported by Fisher et al. [80], to correct for a typographical error in the 1986 paper). Part (a) reproduced with permission from [78]. 1996, Optical Society of America...

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