Fluorescein quantum yield

Many derivatives of fluorescein containing a reactive group at the C-5 position are commercially available [11], Fluorescein isothiocyanate, for example, is widely used as protein tag [12]. These substances have essentially the same spectroscopic properties as the parent compound with the additional capability of binding covalently to proteins. Because of their high emission quantum yields, fluorescein conjugates are extensively used as tracers for microinjection in living cells to gather information on the structure and function of cells, localization of proteins, and cell-to-cell and intracellular diffusion [13-17]. 

C is the concentration of limiting reactant in mol/L, c is the chemiluminescence quantum yield in ein/mol, and P is a photopic factor that is determined by the sensitivity of the human eye to the spectral distribution of the light. Because the human eye is most responsive to yellow light, where the photopic factor for a yellow fluorescer such as fluorescein can be as high as 0.85, blue or red formulations have inherently lower light capacities. 

Calibration. Many approaches have been used to calibrate flow cytometric measurements. Including the comparison of flow and nonflow techniques (radiolabels, spectrofluorometry). In recent years, commercial standards have been introduced which are calibrated in fluorescein equivalents/particle (e.g., 3,000 or 500,000). With labeled ligands, calibration requires determining the relative quantum yield of the ligand compared to pure fluorescein and using the standards to analyze the amount bound on cells. Our ligands (fluorescein isothiocyanate derivatives) are typically 50% as fluorescent as fluorescein. 

One of the interesting features in the structure-photophysical property relationship of fluorescein is that the quantum yield of fluorescein increases under the basic condition. Therefore, many of fluorescein derivatives have been used as pH sensors to measure intracellular pH due to their pH-responding photophysical property [53]. Although fluorescein itself is slightly fluorescent in alcoholic solutions, the addition of alkali (pH > 8) to the fluorescein solution produces the very intense fluorescent alkali salt. The salt form of fluorescein... 

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... 

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. 

Similar results were obtained with the diperoxides 5 (R phenyl) and 5 a (R />-chlorophenyl) and dibenzanthrone or other fluorescers (perylene, rhodamine B, 9.10-diphenylanthracene, anthracene, fluorescein), with quantum yields of the respective chemiluminescence in the range 3.29 X 10 8.... 5.26 X 10 6. 

In 1888, Walter studied the quenching of fluorescence, by the concentration effect, of fluorescein solutions. Nicols and Merrit observed in 1907, in solutions of eosine and resoruflne, the symmetry existing between their absorption and fluorescence spectra. In 1910, Ley and Engelhardt determined the fluorescence quantum yield of various benzene derivatives, values that were still referred to until recent years [18], The works by Lehmann and Wood, around 1910, marked the beginning of analysis based on fluorescence [4],... 

In the literature, fluorescence spectroscopy in OFD has been limited to the use of ultraviolet (UV) or visible dyes as molecular probes.(1) The most common fluorescent dye used in OFD is fluorescein and its derivatives/21 23) Fluorescein possesses a good fluorescence quantum yield and is commercially available with an isothiocyanate functionality for linking to the polymeric support/24-26 Additionally, selective laser excitation can be performed because the absorbance maximum of fluorescein coincides with the 499-nm laser line emitted from an argon laser. Unfortunately, argon lasers are costly and bulky, thus limiting the practicality of their use. Similar difficulties exist with other popular commercial dyes. 

The high molar absorptivities and quantum yields of the large protein fluorophore phycoerythrin (240,000 Da) have been exploited in energy transfer assays. Phyco-erythrin has been used as both donor and acceptor, with several bound antigen molecules per phycoerythrin molecule/86,94) The usefulness of BPE is indicated in competitive assays for human IgG that use fluorescein-labeled antibody as donor to... 

During chase incubation at 4°C, no significant change of the cell-associated fluorescence intensity should be detected over time. Because of the reduced incubation temperature, the metabolism of the cells is minimized, resulting in an inhibition of active uptake processes. Upon subsequent addition of monensin, the fluorescence emission signals should not be altered as well. In doing so, any direct influence of monensin on the quantum yield of the fluorescein label can be excluded [25],... 

Extrinsic fluorescence is used whenever the natural fluorescence of a macromolecule is inadequate for accurate fluorescence measurement. In this case, one can attach a fluorescent reporter group by using the reactive isocyanate or isothiocyanate derivatives of fluorescein or rhodamine, two intensely fluorescent molecules. One can covalently also label a protein s a- and e-amino groups with dansyl chloride (/.e., A,A-dimethylaminonaphtha-lenesulfonyl chloride). Another useful reagent is 8-ani-lino-l-naphthalenesulfonic acid (abbreviated ANS). This compound is bound noncovalently by hydrophobic interactions in aqueous solutions, ANS is only very fluorescent, but upon binding within an apolar environment, the quantum yield of ANS becomes about 100 times greater. 

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...

An important measure of the luminescence is the quantum yield. In effect, this is the probability that a photon will be emitted by the lanthanide given that one photon has been absorbed by the antenna ligand. Since measurement of absolute quantum yields is particularly difficult, the overall quantum yield ( ) is normally measured with reference to certain standards (26) these are routinely [Ru(bpy)3]2+ in water or SulfoRhodamine 101 in methanol for Eu3 +, and quinoline sulfate in 0.1 M HC1 or fluorescein in 1 N NaOH for Tb3+ (27,28). A method has been developed that measures energy transfer from the lanthanide complex to an acceptor of known quantum yield (28). 

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