Fluorescence experimental determination

We have now seen how fluorescence quantum yields and lifetimes are experimentally determined, along with some of the strengths and weaknesses of the methods used. For anthracene these constants have been determined to be... 

Substituting hx = 3.6 cm and K ip/w = K - into Eq. 28 Johnson et al. calculated solute lateral diffusion coefficients in stratum corneum bilayers from macroscopic permeability coefficients. Measurements with highly ionized or very hydrophilic compounds were not performed because of the possible transport along a nonlipoidal pathway. Comparison of the computed Aat values with experimentally determined data for fluorescent probes in extracted stratum corneum lipids [47] showed a highly similar curve shape. The diffusion coefficient for the lateral transport showed a bifunctional size dependence with a weaker size dependence for larger, lipophilic compounds (> 350 Da), than... 

Surface fluorescence of NADH/NADPH can be recorded continuously with a DC fluorimeter and correlated with changes in experimental conditions. A mercury arc lamp (with a 340-375 nm filter in front) is used as a hght source for fluorescence excitation. The fluorescence response of reduced NADH/NADPH was measured at 450-510 nm. The DC fluorimeter and the Hg arc lamp are connected to the kidney by a trifurcated fiber optics light guide. NADH/NADPH fluorescence emission can be corrected for changes in tissue opacity by a 1 1 subtraction of reflectance changes at 340-375 nm from the fluorescence. To determine NADH/NADPH redox state of the total surface area of kidney cortex and to evaluate whether certain areas were insufficiently perfused, fluorescence photographs of the total surface area were taken. The study demonstrated that the surface fluorescence method is simple and provides specific information about the mitochondrial oxidation-reduction state. 

Figure 13. The upper section of the figure is a superposition of an experimentally determined C(f) function for the fluorescing probe 7-(dimethylamino)coumarin-4-acetate ion in the water and biexponential fit of C(t). The lower section in the figure portrays the difference between the experimental C(f) and the biexponential fit on a scale that is expanded by a factor of 3 in the Y direction. From Ref. 33 with permission, from J. Phys. Chem. 93,7040 (1988). Copyright 1988, American Chemical Society.

The rate of diffusion is proportional to the concentration gradient, and the proportionality constant is defined as the diffusion coefficient (D) in Fick s first law of diffusion. Experimental determination of D is commonly performed ex vivo due to the difficulty of measuring concentration gradients in the interstitium. In vivo measurement can be performed in specific tissues, using transparent chamber preparations in combination with the FRAP technique (Berk et al., 1997 Jain et al., 1997 Pluen et al, 2001). However, the in vivo approach is limited only to fluorescent molecules or solutes whose D is not affected by labeling with fluorescent markers. 

Krueger and Weitz [1992] measured the diffusion coefficient of oxygen atoms in the ground 3P state in solid xenon by using the fluorescence of XeO excimers to monitor the 0(3P) concentration. The experimentally determined values are 5.4 x 10-18 and 2.0 x 10 17 cm2/s at 32 and 40 K, respectively. These diffusion coefficients are only about three orders of magnitude smaller than for hydrogen atoms ( 10 14cm2/s at 50 K), as... 

Figure 1 Experimentally determined values of ko obtained employing 3-methylindole as the quencher in homogeneous solvents, plotted as a function of the wavelength of maximum fluorescence intensity (data from Ref. 14). kap values determined in DODAC LUVs ( ) and in dipalmitoyl phosphatidylcholine (DPPC) LUVs (A) have been included. Also are included the experimentally determined value of kap in sodium dodecyl sulfate micelles ( ) and the value of kQ estimated from Eq. (21) ( ).

Figure 1.190 Comparison of simulated and experimentally determined fluorescence turn-off lengths (FTOL) [33] (by courtesy of Kluwer Academic Publishers).

In TPA induced fluorescence spectroscopy the excited state population for fluorescence is created by the simultaneous absorption of two photons instead of the one-photon absorption in standard fluorescence experiments. The principles of this technique are described in Ref. [23]. It allows an experimental determination of two-photon absorption cross-sections provided the material is fluorescent and that its two-photon fluorescence quantum efficiency is known (which is usually assumed to be equal to the one-photon fluorescence quantum efficiency if the same excited state is reached). In this case the method generally provides high sensitivity. 

FP is an alternative readout principle for endopeptidase activity assays. FP or anisotropy measurements allow the detection of changes in the rotational correlation time of particles. These differences in the rotational correlation (or relaxation) time are related to different masses of particles. The experimental determination of steady-state fluorescence anisotropy requires the linear polarization of the light used for the excitation of the probe as well as linear polarization of the emitted fluorescence. Based on data of an appropriate experiment, the fluorescence anisotropy can be calculated as ... 

Pyrene excimer formation still continues to be of interest and importance as a model compound for various types of study. Recent re-examinations of the kinetics have been referred to in the previous section. A non a priori analysis of experimentally determined fluorescence decay surfaces has been applied to the examination of intermolecular pyrene excimer formation O. The Kramers equation has been successfully applied to the formation of intermolecular excimer states of 1,3-di(l-pyrenyl) propane . Measured fluorescence lifetimes fit the predictions of the Kramer equation very well. The concentration dependence of transient effects in monomer-excimer kinetics of pyrene and methyl 4-(l-pyrenebutyrate) in toluene and cyclohexane have also been studied . Pyrene excimer formation in polypeptides carrying 2-pyrenyl groups in a-helices has been observed by means of circular polarized fluorescence" . Another probe study of pyrene excimer has been employed in the investigation of multicomponent recombination of germinate pairs and the effect on the form of Stern-Volmer plots ". 

Many papers have been devoted to the experimental determination of the exciton diffusion constant D. In most of the studies, D was determined by observing how the diffusion of excitons results in their capture by impurities (sensitized fluorescence) or in bimolecular quenching of excitons (reviews of these experiments may be found in (8),(21). The interpretation of such experiments requires that not only the diffusion of the excitons to the acceptor is taken into account, but also the character of the exciton interaction with the acceptor (i.e. with the impurity... 

Many other intermolecular and intramolecular contacts are described by distances (hydrogen bond lengths, van der Waals contact, experimentally determined distances from nuclear Overhauser effect (NOE) spectra, fluorescence energy transfer, etc.) so that the distance matrix representation can be used to specify all the known information about a molecular structure. These bounds are entered into a distance geometry program, as are other bounds that specify constraints on modeling problems, such as constraints to superimpose atoms in different molecules. Hypotheses about intra- or intermolecular conformations and interactions are easily specified with distance constraints models can be built quickly to test different hypotheses simply by changing the distance constraints. 

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