Fluorescent probes characteristics

NHS-rhodamine is an amine-reactive fluorescent probe that contains a carboxy-succinimidyl ester group off the No. 5 or 6 carbons on rhodamine s lower-ring structure (Kellogg et al., 1988). The 5- and 6-isomers are virtually identical in their reactivity and fluorescent characteristics. Similar to TRITC (described previously), NHS-rhodamine can be used to label proteins and other macromolecules that contain primary amine groups. The isomeric forms of the fluorescent probe are available in mixed and purified forms (Invitrogen, Thermo Fisher). The pure forms are... 

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

Antibody molecules can be labeled with any one of more than a dozen different fluorescent probes currently available from commercial sources. Each probe option has its own characteristic spectral signals of excitation (or absorption) and emission (or fluorescence). Many derivatives of these fluorescent probes possess reactive functionalities convenient for covalently linking to antibodies and other molecules. Each of the main fluorophore families contains at least a few different choices in coupling chemistry to direct the modification reaction to selected functional groups on the molecule to be labeled. These choices include amine-reactive, sulfhydryl-reactive, and carbonyl-reactive. Examples of some of the more popular varieties of fluorescent probes can be found in Chapter 9. 

In summary, flow cytometry is clearly useful in evaluating macrophages and their role in toxicity. A major advantage of this technology is the rapid and accurate identification of subpopulations of responding cells from within a mixed population. There is no doubt that the utility of flow cytometry will increase in the future as new fluorescence probes are developed that allow investigators to more clearly assess various macrophage characteristics and the response of these cells to xenobiotics. 

Bucsiova L, Hrdlovil P, Chmela S (2001) Spectral characteristics of fluorescence probes based on pyrene in solution and in polymer matrix. J Photochem Photobiol A Chem 143 59-68... 

In conclusion, lifetimes and quantum yields are characteristics of major importance. Obviously, the larger the fluorescence quantum yield, the easier it is to observe a fluorescent compound, especially a fluorescent probe. It should be emphasized that, in the condensed phase, many parameters can affect the quantum yields and lifetimes temperature, pH, polarity, viscosity, hydrogen bonding, presence of quenchers, etc. Attention should be paid to possible erroneous interpretation arising from the simultaneous effects of several factors (for instance, changes in viscosity due to a variation in temperature). 

The second family ofxanthene dyes is fluorescein and its derivatives. Fluorescein itself is only slightly fluorescent in alcohol solution. In contrast, the alkali salt obtained by addition of alkali exhibits the well-known yellow-green fluorescence characteristic of the fluorescein dianion (uranin). Fluorescein and its derivatives, e.g. eosin Yand erythrosin Y, are known to be very sensitive to pH and can thus be used as pH fluorescent probes (see Chapter 10). 

In Section 3.4, structural effects were often discussed in conjunction with the nature of the solvent. As emphasized in the introduction to this book, the fluorescence emitted by most molecules is indeed extremely sensitive to their microenvironment (see Figure 1.3), which explains the extensive use of fluorescent probes. The effects of solvent polarity, viscosity and acidity deserves much attention because these effects are the basis of fluorescence probing of these microenvironmental characteristics and so, later chapters of this book are devoted to these aspects. The effects of polarity and viscosity on fluorescence characteristics in fluid media and the relevant applications are presented in Chapters 7 and 8, respectively. The effect of acidity is discussed in Sections 4.5 and 10.2. This section is thus mainly devoted to rigid matrices or very viscous media, and gases. 

Fluorescent. These emit light or change their fluorescent characteristics on reacting with cell constituents. (For more details on fluorescent probes etc., see Chapter 3, section 3.5.6)... 

Organizational characteristics of surface-active molecules have been studied by several researchers due to their applications in many areas such as personal care, polymerization, catalysis, drug delivery, separation and purification, enhanced oil recovery and lubrication. The structure of supramolecular organized assemblies formed in different solvents, when a critical concentration is exceeded, determines their properties such as solubilization [1-3], catalysis [1,4-6], adsorption [7-11] and flocculation [12,13]. As such, many techniques have been used to determine their structural properties. In this paper, the results obtained using fluorescence probing for properties of assemblies in solution and at solid-liquid interfaces are discussed in detail after a brief review of relevant assemblies formed by them. 

A suitable fiuorescent probe is an organic molecule, which must change its characteristic parameters with changes in its microenvironment and the parameter must be measurable when the probe is added to the system [54]. The fluorescent probes are categorized as either extrinsic, intrinsic, or covalently bound probes. The intrinsic probes allow a system to be observed without any chemical perturbation. This occurs when the system to be characterized has an in-built fluorescent chromophore unit like tryptophan, tyrosine and phenyl alanine in protein. In some cases the fluorophore is covalently... 

The polarity within a surfactant assembly will be quite different from that of the bulk solution. It is useful to know the micropolarity of these assem-bhes for such applications where different substrates are compartmentalized inside these surfactants. The micropolarity of the surfactant assembhes can be determined using any fluorescence probe whose emission characteristics change with solvent polarity. The emissions of the probe are measured in solvents of known polarities and the polarity of the surfactant assembhes is determined by comparison. 

Eosin emission characteristics depend strongly on the solvent. Specifically transfer from aqueous solution to a nonaqueous solvent shifts the emission of Eosin toward longer wavelengths and increases the emission intensity. Wang and Cheung [142] have used the fluorescence enhancement of the Eosin label to study the association of troponin I with troponin C. Similarly Skou and Esmann [143] and Helmich de Jong et al. [144] have used Eosin itself as a fluorescent probe to study the conformational changes of enzymes involved in ionic transport. 

Many of the present models used to describe fluid-solid phase equilibria require one to assume that the solute is at infinite dilution. That is, researchers have often assumed that solute-solute interactions are nonexistent. Recently, Brennecke et al. used the fluorescent probe pyrene to investigate the possibility of solute-solute interactions in C02, C2H4, and CF3H (7-9). Pyrene is an interesting probe because it can form a characteristic excited-state dimer (excimer) during its excited-state... 

Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, by Richard P. Haugland. Molecular Probes (http // www.molecularprobes.com/) makes a vast range of fluorescent chemicals that are useful in flow cytometric analysis. The website and the paper or CD Handbook provide a great deal of information about the use of these chemicals as well as about their photochemical characteristics. A required reference book for every flow cytometrist. 

The ESIPT of 2-(2 -hydroxyphenyl)-4-methyloxazole (HPMO) (27) has been explored by Douhal and co-workers [166] for its probe characteristics in a variety of organized media which include cyclodextrin, calixarene, micelle, and HSA. The incorporation of HPMO into hydrophobic cavities in an aqueous medium involves the rupture of its intermolecular hydrogen bond to water and formation of an intramolecular hydrogen bond in the sequestered molecule. Upon excitation (280-330 nm) of this entity, a fast intramolecular proton-transfer reaction of the excited state produces a phototautomer (28), the fluorescence of which (Xm = 450 170 nm) shows a largely Stokes-shifted band. Because of the existence of a twisting motion around the C2—C bond of this phototautomer, the absorption and emission properties of the probe depend on the size of the host cav-... 

A recent paper by Chauret et al. described the discovery of a novel fluorescent probe that is selectively metabolized by CYP3A in human liver microsomes (32). This probe, DFB [3-[(3,4-difhiorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl) phenyl] furan-2(5F/)-one], is metabolized to DFH [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5//)-one], which has fluorescent characteristics (Fig. 7). In vitro CYP reaction phenotyping studies (cDNA-expressed CYP proteins and immunoinhibition experiments with highly selective anti-CYP3A4 antibodies) demonstrated that DFB was metabolized primarily by CYP3A4 (Fig. 8). Furthermore, metabolism studies performed with human liver microsomes obtained from different donors indicated that DFB dealkylation and testosterone 6P-hydroxylation correlated well (Fig. 9). 

Their emission bands that excited at the region away from the photochromi-cally active absorption bands could be conveniently regulated in a reversible manner by the photoisomerization of the dithienylethene moieties. This characteristic was useful for the application in fluorescence probes and non-destructive readout for erasable memory media. 

Some lanthanide ions when complexed with UV-absorbing ligands, can efficiently accept energy from the excited state of the ligand and produce highly enhanced emission characteristics of the metal ion. Rare earth complexes have some advantages over organic fluorescent probes such as fluorescein, rhodamines, umbelliferones such as... 

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