Steady-state fluorescence methods

This is a steady-state competitive method, applicable when a solute is capable of fluorescing. We consider the simplest case. The solute A undergoes excitation to the excited singlet state A upon absorption of radiation of frequency... 

Situation with H-bonding also demands to take into account the fact that alcohols have ability to form various associates or even clusters at normal conditions. The most efficient method for determination of inhomogeneity in the excited states is fluorescence polarization measurements. These methods also frequently applied for studying of solvent viscosity, they may be provided in two variants steady state and time-resolved. Relations for time-resolved and steady state fluorescence anisotropy may be given as [1, 2, 75] ... 

Steady-state fluorescence spectra, fluorescence quantum yield (F) and lifetimes (tf) of DTT 15 and DTP 23a were estimated as shown in Table 8. F for DTT is higher than DTP. F for DTP is very small and it was difficult to estimate an accurate fluorescence lifetime by the photon counting method due to weak fluorescence. It is noted that the for DTP depends largely on the solvent and is 7.7 x 10-5 in acetonitrile. This low F value has been attributed to an addition reaction with the solvent. 

Domingo, B., Sabariegos, R., Picazo, F. and Llopis, J. (2007). Imaging FRET standards by steady-state fluorescence and lifetime methods. Microsc. Res. Tech. 70, 1010-21. 

Methods. Absorption spectra were recorded using an Hitachi model 150-20 spectrophotometer/data processor system. Uncorrected steady-state fluorescence emission spectra were recorded using a Perkin-Elmer MPF-44A spectrofluorimeter. These spectra were collected and stored using a dedicated microcomputer and then transferred to a VAX 11/780 computer for analysis. Fluorescence spectra were corrected subsequently for the response characteristics of the detector (21). Values of the fluorescence quantum yield, <) , were determined relative to either quinine bisulfate in IN H2S04 )>f =... 

The dipole-dipole interactions of the fluorophore in the electronic excited state with the surrounding groups of atoms in the protein molecule or with solvent molecules give rise to considerable shifts of the fluorescence spectra during the relaxation process. These spectral shifts may be observed directly by time-resolved spectroscopic methods. They may be also studied by steady-state spectroscopic methods, but in this case additional data must be obtained by varying factors that affect the ratio between tf and xp. 

In systems where only dynamic quenching occurs, then steady-state fluorescence intensities can be measured instead of lifetimes/101 103-,07) In experiments where comparisons are being made (i.e., for a comparison of different experimental conditions or types of membrane), it is important that the lifetime of the fluorophore (r0) is not affected by the experimental conditions. Fluorescence intensities can be obtained much more rapidly and without specialized instrumentation. Blatt and Sawyer(101) have employed a relationship essentially the same as Eq. (5.20) in this way. They have pointed out that since the quenching mechanism is collisional, the partition coefficient that is derived is a partition coefficient of the quencher into the immediate environment of the fluorophore and is therefore a local Kp. It is therefore possible to investigate the partition coefficient gradient across the lipid bilayer by using a series of probes, such as the anthroylstearates,(108) located at different depths. In their method, Eq. (5.20) has the form... 

M. F. Blackwell, K. Gounaris, S. J. Zara, and J. Barber, A method for estimating lateral diffusion coefficients in membranes from steady-state fluorescence quenching studies, Biophys. J. 51, 735-744 (1987). 

Methods for the measurement of absolute quantum yields have been reviewed. 6 In most steady-state fluorescence experiments relative fluorescence quantum yields are determined by comparison with appropriate standards (see Section 7.7.1.2.1). 

Turro and Yekta [142] discussed a simple method for measuring aggregation number by steady-state fluorescence. In this method, the decrease of the fluorescence intensity of a micelle-bound probe is monitored as a function of the quencher concentration and is fitted to the equation [143]... 

Steady-state fluorescence spectra are obtained for dilute solutions of 16 aromatic polyesters with the repeating unit ABm. The chromophore, A, is isophthaloyl or phthaloyl, and the flexible spacer, B, Is methylene or oxymethylene. The number of methylene units Is 2 - 6, and the number of oxyethylene 1 - 4. Results obtained are interpreted within the frame of the RIS method. 

Figure 12 Conformational transition of BpUreG as revealed by steady-state fluorescence signals, (a) Steady-state emission spectra of BpUreG at 24 C at increasing concentrations of GuHCI (from 0 M to 3 M, incubation time of 10 min), (b) Changes in emission max (black circles) and steady-state anisotropy (clear squares) as a function of denaturant concentration. The solid lines represent the fits by a nonlinear least-squares method of the experimental data. (Reprinted from Reference 187 with permission of the ACS.)...

We have used the spectral reconstruction method to obtain a time-resolved fluorescence spectrum [13] When the fluorescence up-conversion method is used, the relative intensity between each wavelength becomes uncertain because the angle of the nonlinear crystal has to be tuned at each wavelength of observation. However, the intensity of the fluorescence /(A, (), at a given time t and wavelength A, can be obtained from the normalized fitted decay series D(t, A) and intensity of the steady-state fluorescence /0(A) ... 

The theory of rotation effects on prolate luminescent molecules in solution and its experimental verification have been developed and compared. Generalized diffusion equations for the rotational motion of an asymmetric rigid motor have been used to given an expression for steady-state fluorescence depolarization. " The radiationless transition from the first excited singlet state of Eosin has been measured by optoacoustic relaxation, and the absolute fluorescence quantum yields of organic dyes in poly(vinyl alcohol) have also been measured by the photoacoustic method. The accuracy of the method has been discussed in the latter paper. Actinometry in flash photolysis experiments has been assisted by new measurements on the extinction coefficient of triplet benzophenone. Matrix-isolation fluorescence spectrometry has been used to detect polycyclic aromatic hydrocarbons from gas chromatography. ... 

Emission spectra and absorption spectra were recorded on a Perkin-Elmer 650-iOS Fluorescence Spectrophotometer and a Perkin-Elmer 320 tn/ Spectrophotometer, respectively. Fluorescence decay data were obtained on a single-photon-counting apparatus from Photochemical Research Associates. The samples were bubbled witli nitrogen for the steady-state fluorescence spectra and the fluorescence decay measurements. In some cases, front face spectra were taken. The data were analyzed by a software package from PRA based on the iterative convolution method. NMR spectra were obtained on a JEOL FX90Q, and FTIR spectra were recorded on a Nicolet 5DX. The elemental analyses were conducted by M-H-W Laboratories of Phoenix, AZ. 

In certain cases of well-behaved photoacids, typically having piT values in the range of 0-3, Forster cycle predictions may be tested against piT values found by two direct experimental methods. The first is titration of the excited photoacid with a stronger mineral acid until an end-poinf is reached [9]. The titration of the excited photoacid is monitored by following the change in the relative quantum yield of the photoacid or the photobase as a function of the solution pH. Taking the steady-state fluorescence spectra of the photoacid at each titration point is... 

Arguably, the first evidence for the general validity of the pK scale came from steady-state fluorescence titrations of well behaved photoacids such as 2-naphthol [27]. As already indicated, this method was largely developed by Weller [9] and resulted in IC values which were in general agreement with the Forster cycle predictions (see below). 

Despite the errors that can arise in both the Forster cycle and the steady-state fluorescence the pAT obtained by both methods usually agree to within 1 or 2 pK units (see tables in Ref. 28). For triplet states appUcation of either method is often difficult or even impossible. Instead it is necessary to use a kinetic approach. The amounts of conjugate acid and base in the triplet state are followed by time-resolved excited-state absorption spectroscopy. ... 

The steady-state fluorescence anisotropy/polarization method is also simple and relies on the fact that the probe molecule will tumble rapidly in solution when free, but will have restricted motion upon binding to a macromolecule. Optical excitation of the probe by polarized light will result in preferential absorption by those molecules whose absorption transition dipole is parallel to the electric field vector direction of the light. The subsequent fluorescence will be partially polarized. The definitions of anisotropy (r) and polarization (P) are [188]... 

Most current assays use immunofluorescence or GFP fluorescence to evaluate the nucleocytoplasmic distribution of endogenous nuclear proteins (Corbett et al., 1995 Lim et al, 1995 Schlenstedt et al, 1995) or NLS-containing reporter proteins (Nehrbass et al, 1993 Schlaich and Hurt, 1995) in the steady state. These methods are usually sensitive only to defects that severely impair the transport apparatus (see example below). Even the use of inducible promoters to express reporter proteins after shifting cells to nonpermissive conditions (Corbett et al, 1995 Schlenstedt et al, 1995) requires at least 30-60 min to produce an adequate signal and is, therefore, often too slow. A clever assay was recently described to measure defects in nuclear export signal-mediated protein export in yeast (Lee et al, 1996). An attractive in vivo approach now in use in tissue culture cells is the application of hormone-stimulated GFP-glucocorticoid receptor import to study import kinetics (Htun et al, 1996 Carey et al, 1996). 

In the preceding ch t we described the measurement and interpretation of steady state fluorescence anisotropies. These values are measured using continuous illumination and r resent an average of the anisotropy decay ov - the intensity decay. Measurement of steady-state anisotropies is simple, but interpretation of the steady-state anisotropies usually d nds on an assumed form for the anisotropy decay, which is not directly observed in the experiment. Additional information is available if one measures the time-dependent anisotropy> that is, the values of r(t) following pulsed excitation. The form of the anisotropy decay depends on the size, shape, and flexibility of the labeled molecule, and the data can be compared with the decays calculated from various molecular models. Anisotropy decays can be obtained using the TD or the FD method. 

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