Fluorescence intensity, fluorescein

Note The full fluorescence intensity usually only develops about 30 min after the dipping process it then remains stable for several days if the chromatograms are stored in the dark (1, 5]. Fluorescein sodium can be employed in the reagent in place of 2, 7 -dichlorofluorescein [5]. The detection limits lie in the lower nanogram to picogram range [1, 5]. 

Fig. 16 Contours of fluorescent intensity in frozen sections of the rabbit eye following 15 pL injection of marker solution in the central vitreous cavity injection was conducted through the superior rectus muscle 15 hours following injection of 0.2% sodium fluorescein 14 days following injection of 0.1% FITC-dextran, molecular weight 66,000. (From Ref. 230.)... 

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... 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

Another approach of energy transfer-based probe has been demonstrated for a-Amylase sensing/98 In this case amylose was doubly labeled, by fluorescein derivative (donor) and Procion Red MX8B (acceptor). As a-Amylase catalyzes the cleavage of the amylose into smaller units, the average distance between fluorescein and Procion Red increases, which reduces the degree of quenching. The rate of increase in fluorescence intensity is proportional to ee-amylase activity. 

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],... 

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. 

Fluorochromes have been introduced that offer excitation and emission spectra similar to those of fluorescein, but that overcome some of fluorescein s limitations. BODIPY FL has a short Stokes shift, but offers higher fluorescence intensity, and is claimed to be more photostabile and less pH-sensitive than fluorescein. Oregon Green 488 and the newly introduced Alexa 488 fluorochromes have spectra nearly identical to those of fluorescein, but are considerably more photostabile, and produce less quenching of fluorescence with higher numbers of fluorochromes per antibody than does fluorescein. 

Rhodamine conjugates are less sensitive to pH and are less prone to photohleaching than are those of fluorescein. Their fluorescence intensity is generally lower than that of fluorescein conjugates under comparable conditions of excitation, but the intense 546-nm excitatory light provided by the mercury lamp of a fluorescence microscope may make rhodamine appear brighter (7). 

A limitation of the flow cytometric binding assay has been the precise determination of the receptor affinity and calculation of the receptors per cell. This limitation appears to have been overcome by the development of fluorescein and phycoerythrin compensation-calibration standards (Flow Cytometry Standards Corp., Research Triangle Park, NC). These standards have made it possible to quantify the fluorescence intensity of samples labeled with fluorescein or phycoerythrin, and relate the intensity to molecules of equivalent soluble fluorochrome. These standards have been utilized in quantitative studies of neutrophil chemoattractant-ligand interaction (4). 

Fig. 7 Fluorescence intensity of the PNA/DNA hybrid vs. separation temperature. Fluorescein-labeled PNA probes with complementary 3 sequence. Voltage 20 kV. Detection LIF 488/520 nm. Buffer IX TBE/30% formamide (pH 8.3). The following M13 probes were used 5 -fluorescein-00-TTT TCC CAG TCA CGA (perfect match), 5 -fluorescein-OO-TTT TCC CAG GCA CGA (single mismatch), 5 -fluo-rescein-OO-TTT TCA CAG GCA CGA (double mismatch). (From Ref. 37.)...

Fluorometric methods have been developed for determining the concentrations of more than 50 elements in the periodic table. These methods depend on the measurement of changes in the fluorescence intensity of a fluorescent dye on interaction with the species to be analysed. The concentration of the substance being analysed is proportional to the fluorescence intensity, determined from calibration curves. The interactions can take the form of ionic associates between a dye cation and a metal complex anion, e.g. AgBrj with a rhodamine cation, or alternatively with a fluorescent dye anion, e.g. fluorescein and a complex cation. In another method, the changes... 

The xanthenes exist in solution in several different forms depending on pH, as shown in Figure 2 and Table 1 [18]. The emission quantum yield of fluorescein depends on the acidity of the solution, the fluorescence intensity decreasing as the protonated forms of the dye come to predominate with decreasing pH. This pH sensitivity allows fluorescein derivatives to be employed as pH indicators, to measure the pH inside living cells [19-22], at water-lipid interfaces [23], and in the interior of phospholipid vesicles [24]. The sensitivity of fluorescein emission to the pH of the medium has also been used to measure lateral proton conductances at water-lipid interfaces [25-28] and proton translocation across phospholipid vesicles [29] and to determine the electrostatic potential of macromolecules [30, 31]. The pheno-... 

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