Fluorophore saturation

The intrinsic sensors are based on the direct recognition of the chemicals by its intrinsic optical activity, such as absorption or fluorescence in the UV/Vis/IR region. In these cases, no extra chemical is needed to generate the analytical signal. The detection can be a traditional spectrometer or coupled with fiber optics in those regions. Sensors have been developed for the detection of CO, C02 NOx, S02, H2S, NH3, non-saturated hydrocarbons, as well as solvent vapors in air using IR or NIR absorptions, or for the detection of indicator concentrations in the UV/ Vis region and fluorophores such as quinine, fluorescein, etc. 

Cyanines have been widely used as laser dyes, and as saturable absorbers in modelocked and Q-switched laser systems. 8, 50) The propensity of most cyanines to photooxidize which makes them useful in photographic film and as saturable absorbers makes them less than desirable as fluorophores in other applications. The use of... 

The excitation spectrum of bound ethidium bromide published by Molecular Probes (Figure 12.5) indicates that, at saturation, fluorophore in the presence of DNA does not absorb in the region between 400 and 420 nm. Also, we can see from the absorption spectra of ethidium bromide recorded at different concentrations of DNA (Figure 12.3) that the OD... 

The conception of GSD microscopy to reversibly switch the fluorophore to a metastable state has led to the consideration of molecular switches between states of even longer lifetimes. As a matter of fact, the ultimate saturable or switching transition occurs between two stable states [38-41]. The advantage of switching between two stable states is obvious since there are no spontaneous interstate transitions, it follows that Ig —> 0. As a result, it should be possible to implement huge values Imax/Is which, following (19.1), should yield very small Ar even at low /max-... 

Fig, 3.32 exhibits the normalized fluorescence emission spectra of calcofluor free in phosphate-NaCl buffer (b), in the presence of a saturated concentration of a i-acid glycoprotein (c) and of HSA at a concentration close to saturation (a). The emission maximum of the fluorophore shifts from 435 nm in buffer to 448 or to 415 nm, respectively, in the presence of a i-acid glycoprotein or HSA. The red shift (13 nm) of the maximum observed in presence of a i-acid glycoprotein compared with the maximum obtained in water indicates that the microenvironment of the excited state of calcofluor on a i-acid glycoprotein is hydrophilic. The blue shift (20 nm) observed in presence of the serum albumin corresponds to an emission from a hydrophobic environment. Thus, the interaction of calcofluor with serum albumin arises from molecular energy transitions different from those present in the interaction between the fluorophore and a i-acid glycoprotein. 

The fluorescence signal from fluorophores of complex organic compound (COC) under powerful laser excitation is represented as the nonlinear function of the number of detected fluorescence photons Nh (or fluorescence intensity In) on the photon fluxes F of pumping radiation (Filipova et al., 2001). The dependence Nn(F) is called fluorescence saturation curve, its typical view is represented in the Fig. 1(a). There are several reasons for that nonlinear dependence the non-zero lifetime of orj nic molecules in excited state intercombination conversion intermolecular interactions including singlet-singlet annihilation, etc. 

The parameters of saturation curves depend on photophysical characteristics of molecules fluorophores, so that such characteristics can be extracted from these curves after resolving an inverse problem (Fadeev et al., 1999). This is a basement of nonlinear laser fluoiimetiy as a method for investigation of photophysical properties of CCXI. To solve the inverse problem, we should first calculate (either analytically or numerically) the theoretical saturation curves by using the fluorescence response formation model of an ensemble of fluorescent molecules under their excitation by laser radiation. In present work two models have been used the conventional model of fluorescence response formation and the model of localized donor-acceptor (LDA) pairs. 

As was mention above, the photophysical parameters of fluorophores (o> K sz and r in the model (la) and Td, ta, Od, Oa, Kda and Kss in the model (lb)) can be determined from the dependence Nn(F), by solving the inverse problem. However, in experiments, it is convenient to normalize the number of detected fluorescence photons Nn to the reference signal (will denote as Nfe/), which can represent a part of exciting radiation directed to the reference channel of the detection system by a beamsplitter or a Raman scattering signal from water molecules (Fadeev et al., 1999). In this case, one has to deal with the dependence [(F)]- =NRe/Nn (which is also called a saturation curve, (F) is the fluorescence pjarameter) rather than Nfi(F). According to the practical experience such normalization also helps to increase the stability of the inverse problem solution. In the absence of saturation, 0 stop ... 

Samples were prepared by exposing the silica gel, previously dried at 150°C for 24 h, to either cyclohexane or pentane solutions ctmtaining selected amounts of the fluorophore. The solvent was carefidly removed under vacuum when required. Complete probe adsorption of liquid-solid samples was verified with absorption spectroscopy. Less than 0.07% of the silica surface was typically covered by the probe. Immediately before data collection, dry samples were evacuated under vacuum at 125-130°C for 30 min. The total dehydration procedure was sufficient to remove the physisorbed water while leaving the surface silanol functionality intact [12]. Selected amounts of oxygen were introduced into a constant-position sample call by a series of stopcock manipulations and a vacuum line. Liquid-solid samples were deoxygenated ([O2]final < 10 M) by bubbling the samples with solvent-saturated nitrogen for 30 min. 

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