Environment-sensitive lifetimes

Fluorescence lifetime-based applications require probes and labels with environment-sensitive lifetimes, while immunoassays or hybridization-based analysis require fluorescent tracers preferably labeled with a single, mono-reactive fluorescent label. 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Due to their low sensitivity toward the environment, cyanine dyes are perfect candidates as fluorescent labels. Squaraine dyes on the other hand display a highly environment-sensitive response and are therefore not only useful as fluorescent probes and labels but also, in particular, well-suited for lifetime-based applications. 

In conclusion, this review illustrates the last decade (2001-2012) of research endeavour devoted to the sensitisation of lanthanide luminescence by a nonlinear two- (multi-) photon process. The motivation for this project is to combine the unique spectroscopic properties of lanthanide complexes (sharp transitions, long luminescence lifetime, environment sensitivity, circularly polarised luminescence, etc.) with the huge potentialities of the biphotonic excitation (NIR excitation, 3D resolution, etc.), in particular for bio-imaging applications. To that end, several scientific and instmmental breakthroughs have been performed almost simultaneously by several groups around the world ... 

Diatomic molecules have only one vibrational mode, but VER mechanisms are paradoxically quite complex (see examples C3.5.6.1 and C3.5.6.2). Consequently there is an enonnous variability in VER lifetimes, which may range from 56 s (liquid N2 [18]) to 1 ps (e.g. XeF in Ar [25]), and a high level of sensitivity to environment. A remarkable feature of simpler systems is spontaneous concentration and localization of vibrational energy due to anhannonicity. Collisional up-pumping processes such as... 

It may be felt that the initiation of a stress-corrosion test involves no more than bringing the environment into contact with the specimen in which a stress is generated, but the order in which these steps are carried out may influence the results obtained, as may certain other actions at the start of the test. Thus, in outdoor exposure tests the time of the year at which the test is initiated can have a marked effect upon the time to failure as can the orientation of the specimen, i.e. according to whether the tension surface in bend specimens is horizontal upwards or downwards or at some other angle. But even in laboratory tests, the time at which the stress is applied in relation to the time at which the specimen is exposed to the environment may influence results. Figure 8.100 shows the effects of exposure for 3 h at the applied stress before the solution was introduced to the cell, upon the failure of a magnesium alloy immersed in a chromate-chloride solution. Clearly such prior creep extends the lifetime of specimens and raises the threshold stress very considerably and since other metals are known to be strain-rate sensitive in their cracking response, it is likely that the type of result apparent in Fig. 8.100 is more widely applicable. 

The lifetime detection techniques are self-referenced in a sense that fluorescence decay is one of the characteristics of the emitter and of its environment and does not depend upon its concentration. Moreover, the results are not sensitive to optical parameters of the instrument, so that the attenuation of the signal in the optical path does not distort it. The light scattering produces also much lesser problems, since the scattered light decays on a very fast time scale and does not interfere with fluorescence decay observed at longer times. 

A major advantage of fluorescence as a sensing property stems from the sensitivity to the precise local environment of the intensity, i.e., quantum yield (excited state lifetime (xf), and peak wavelength (Xmax). In particular, it is the local electric field strength and direction that determine whether the fluorescence will be red or blue shifted and whether an electron acceptor will or will not quench the fluorescence. An equivalent statement, but more practical, is that these quantities depend primarily on the change in average electrostatic potential (volts) experienced by the electrons during an electronic transition (See Appendix for a brief tutorial on electric fields and potentials as pertains to electrochromism). The reason this is more practical is that even at the molecular scale, the instantaneous electric... 

The UV-visible absorption and emission spectra and excited state lifetimes of polymers are sensitive to chemical structure, polymer conformation and molecular environment and thus information concerning these properties is accessible by electronic spectroscopy measurements (4-6). One example of the application of such measurements is given in Figure 3 which illustrates the possible energy dissipation pathways which can occur in a polymer containing aromatic side groups following absorption of radiation. 

The chromophore environment can affect the spectral position of the absorption and emission bands, the absorption and emission intensity (eM, r), and the fluorescence lifetime as well as the emission anisotropy, e.g., in the case of rigid matrices or hydrogen bonding. Changes in temperature typically result only in small spectral shifts, yet in considerable changes in the fluorescence quantum yield and lifetime. This sensitivity can be favorably exploited for the design of fluorescent sensors and probes [24, 51], though it can unfortunately also hamper quantification from simple measurements of fluorescence intensity [116], The latter can be, e.g., circumvented by ratiometric measurements [24, 115],... 

Montalti and co-workers studied dansyl [27] and pyrene [28] derivatives and found the fluorescence quantum yields and excited-state lifetime of these two dyes increased in DDSNs. They attributed the enhancements to the shielding effect from the quenchers or polar solvent in the suspension. Their studies also demonstrated that the lifetime of the doped dye molecules was also dependent on the size of the DDSNs. Small DDSNs had a larger population of the short-living moieties that were more sensitive to the environment outside the DDSN. In contrast, the large DDSN had a larger population of the long-living moieties that were not sensitive to the environment. 

The behavior of practically all luminescent materials is sensitive to various parameters of physical and chemical origin. The excited state lifetimes and average intensities of the fluorescence and/or phosphorescence of these materials are modulated, for example, by temperature, oxygen, pH, carbon dioxide, voltage, pressure, and ionic strength. Consequently, the luminescence of various materials could be used, in principle, to monitor parameters of interest in medicine, industry, research, and the environment. 

Phosphorescence is readily detectable from most types of proteins at room temperature. Tryptophan phosphorescence lifetimes and yields are very sensitive to environment, and therefore phosphorescence is sensitive to conformational changes in proteins. Fundamental questions concerning exactly what parameters affect lifetime and spectra of tryptophan in proteins remain still to be answered. 

The fluorescence lifetime is sensitive to the environment of the fluorophore, and in membranes this usually means the surrounding fatty acyl chains or the membrane protein interfacial region (see summary in Table 5.3). Generally, the lifetime of membrane-bound fluorophores is rather less sensitive to the types of subtle alterations which are encountered in membranes as compared to the fluorescence anisotropy parameters. The gel-to-liquid crystalline phase transition is a notable exception where most fluorophores show an alteration in lifetime properties. Although, again, the anisotropy (see below) is the most sensitive parameter in this regard, the fluorescence lifetime has been used with considerable success in the study of phase transitions and lateral phase separations. Fluorophores used to yield information on the... 

The fluorescence emission maximum, quantum yield, and lifetime of a fluorophore are very sensitive to its immediate environment. A blue shift in the emission maximum and an increase in the fluorescence quantum yield or lifetime is generally observed when a fluorophore is transferred form a polar solvent to a nonpolar one or when it binds to a hydro-phobic protein site. Furthermore, fluorescence quenching or enhancement may result from interactions of the fluorophore with various structural elements in its vicinity. 

The enormous cost of multiple-species, multiple-dose, lifetime evaluations of chronic effects has already made the task of carrying out hazard assessments of all chemicals in commercial use impossible. At the same time, quantitative structure activity relationship (QSAR) studies are not yet predictive enough to indicate which chemicals should be so tested and which chemicals need not be tested. In exposure assessment, continued development of analytical methods will permit ever more sensitive and selective determinations of toxicants in food and the environment, as well as the effects of chemical mixtures and the potential for interactions that affect the ultimate expression of toxicity. Developments in QSARs, in short-term tests based on the expected mechanism of toxic action and simplification of chronic testing procedures, will all be necessary if the chemicals to which the public and the environment are exposed are to be assessed adequately for their potential to cause harm. 

Phase sensitivity A phase-sensitive flow cytometer quantifies the life time of the fluorescence emitted by particles. The decay time of fluorescence from a given fluorochrome is altered by changing chemical environments, and therefore measurement of fluorescence lifetimes can provide information about the microenvironment surrounding the fluorescent probe. 

The exceptionally long lifetime of Ln3+ ion excited states (0.1-2 ms) might be expected to render Ln3+-sensitization schemes susceptible to quenching [consider Eq. (3) for large x0] however, this is not the case. Buried deep within the ion, f-centered orbitals are shielded from the external environment... 

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