Fluorescence anisotropy parameter

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 anthroylstearate series of fluorescent probes can be used to give information on lipid dynamics at different depths into the lipid bilayer.( 74) Compared with DPH, the anthroylstearates are less fluorescent and higher probe lipid ratios (1 100 compared to <1 400 with DPH) are needed. Although it is possible to extract time-resolved fluorescence anisotropy parameters with the anthroylstearates, so far studies have been largely confined to model lipid bilayers.(8 75 76)... 

The region at the surface of membranes and the underlying phospholipid head-group region are of particular interest since these regions can vary considerably with variations in the membrane phospholipid composition and under the influence of external molecules such as ions and hydrophobic molecules. The fluorescence anisotropy parameter tends to be less useful for examining this region since it is already intrinsically disordered and the... 

Fluorescence depolarization time courses contain information about rotational diffusion (26). The main difficulty with streak camera measurements of time-resolved fluorescence depolarization is limited signal-to-noise ratio of single shot records. The fluorescence anisotropy parameter is defined as s... 

Table 15J Fluorescence anisotropy parameters for labelled acrylic polymers...

FIG. 11 Order parameter variation along acyl chains in red cell ghosts ( ), small unilamellar vesicles of egg phosphatidylcholine (V), and paraffin oil (+), as determined by the fluorescence anisotropy decay of the w-anthroyloxy fatty acid probes. (Reprinted by permission from Ref. 12.)... 

The diffusion constant D with the underlying microviscosity , and the two order parameters , <(P4> reflecting the degree of orientational constraint have been successfully determined from the fluorescence anisotropy decay in... 

The curves are broadened in the near-field range for high g-values which are found very often in biological tissues (see lower curve that has been calculated for a typical tissue anisotropy ofg = 0.95). In all cases, the fluorescence diffuses much wider than the primary radiation but as far as we know no exact values are available for high anisotropy parameters. 

Besides the fluorescence coefficient, the optical absorption and scattering coefficients of the sample are the most important parameters in quantative fluorescence spectroscopy of turbid media. In principle two or, if the anisotropy parameter has to be determined, three independent measurements are sufficient to separate the coefficients that appear in all equations as sums or proportions. However, for better accuracy, one of the geometrical parameters (sample thickness, angle of incidence, distance from the irradiated spot) as well as the wavelength of irradiation should be varied over a wide range, and then the data should be fitted with the help of the corresponding model equation. 

The first decision to be made in designing an experiment to measure the motional properties of membrane lipids concerns the type of probe molecule. Too often, this choice is made from the point of view of convenience or tradition rather than suitability, although there is now a considerable range of suitable fluorophores from which to choose. The second consideration is the type of measurement to be made. The most detailed and complete motional information is obtained from a time-resolved fluorescence anisotropy measurement which is able to separate the structural or orientational aspects from the dynamic aspects of fluorophore motion. Steady-state anisotropy measurements, which are much easier to perform, provide a more limited physical parameter relating to both of these aspects. 

Since steady-state data are much easier to obtain, some effort has been directed to methods for deriving time-resolved anisotropy parameters from the steady-state anisotropy/2 4549-1 A number of relationships have been described, some of which require knowledge of r0 and the fluorescence lifetime (see, e.g., Ref. 48). An example(50) of such an empirical relationship is... 

Extensions of the analysis of time-resolved fluorescence anisotropy decay data in terms of two order parameters have also been developed (see, e.g., Refs. 51-54). Thus, the corresponding higher order parameter term is <7%) given by(53)... 

H. Pottel, W. van der Meer, and W. Herreman, Correlation between the order parameter and the steady-state fluorescence anisotropy of l,6-diphenyl-l,3,5-hexatriene and an evaluation of membrane fluidity, Biochim. Biophys. Acta 730, 181-186 (1983). 

As we explained in the previous section, fluorescence decays do not bring any direct evidence about energy transfer among DNA bases within a helix. In contrast, fluorescence anisotropy decays can provide this type of information. Such a possibility is based on the correlation of macroscopic observables to molecular parameters. On the molecular scale, r is related to the angle 6 formed between the transition dipoles associated to photon absorption and photon emission ... 

Jablonski showed in 1960 that the fluorescence depolarization can be described naturally by another parameter, the fluorescence anisotropy r, defined by ... 

Order parameters are used to interpret data on order and fluidity of a number of probes in lipid membranes obtained by measurements of fluorescence anisotropy decay 32 Ambiguities in the interpretation of time resolved fluorescence anisotropy measurements in lipid vesicle systems with DPH or TMA-DPH probes are attributed to the unsatisfactory models being used to interpret the data . The solubilisation of diphenylpolyenes in lipid bilayers has been critically examined33. It is concluded that such probes are satisfactory if used at low concentrations. 

TABLE 1.4 Fit Parameters of the Fluorescence Anisotropy Decays Measured for m-C1P (x —1-4) in Toluene with Xexc — 488 and /.nu = 600 nm at Which There Is Only Monomer Emission and Average Chromophore-Chromophore Distances (tfDA)... 

The problem of pure lifetime identifieation is that the lifetime differences are often relatively small. Moreover, there may be some variation in the lifetime of a single type of molecule due to different loeal environments. It is therefore useful to add more parameters to the identifieation algorithm. By using the fluorescence anisotropy, rhodamine 133 and EYFP were identified with only 1% misclassifica-tion [442]. 

Figure 11,22 Two-photon STED. The sample is excited with a ca. 100 fs laser pulse (PUMP). A time-delayed picosecond laser pulse (DUMP) is used to partially deplete the exdted state population. This causes an abrupt change in fluorescence polarization due to the preferential removal of molecules oriented parallel to the polarization direction of the PUMP. Analysis of the anisotropy change shows that it is sensitive to both the initial (pre-dumped) fluorescence anisotropy (/ u) and the pre-dumped excited state value of (a4o)- This order parameter is unobservable in conventional fluorescence experiments...

A detailed evaluation of the structural parameters affecting the photophysical properties was performed for the didodecyloxy-substituted quinquephenyl rigid-flexible polyethers 60. More particularly, the odd-even effect was observed for dilute polymeric solutions by means of steady-state and time-resolved fluorescence anisotropy. The different orientations of the quinquephenyl chromophores, and subsequently of their luminescent dipoles, concerning the polymers with an odd number of methylene units x=7, 9, 11) and those with an even (x = 8, 10, 12) one, were found responsible for the observed strong deviations in frozen and dilute solutions, where the flexible aliphatic chains are forced to adopt a nearly sta ered, lower energy conformation. 

Figure 4. Order parameters S of chlorophyll a in DGDG membranes (1 250) as a function of the wavelength of excitation, as found from linear dichroism experiments. The continuous line is a theoretical fit based on fluorescence anisotropy measurements in castor oil.

As can be seen from table 1, the order parameters can be equally well obtained from LD as from AFD measurements. The table also gives the calculated value of obtained from a fit to the values of r and TC2f the fluorescence anisotropy measured at the two respective emission wavelengths of chlorophyll a (3). 

That is, the observed r value depends not only on the r, value and mole fraction, X(, for the N and U states, but r also depends on the quantum yield of each state, i. If one of the states has a larger quantum 3deld than the other, the result will be a skewing of the plot of r versus perturbation axis toward the more dominant fluorescing state. In principle, if the Op (or relative intensity) of the native and unfolded states are known, one can still use r values to obtain thermodynamic information. We have shown elsewhere that plots of fluorescence anisotropy versus denaturant concentration for the unfolding of a protein can give the same thermodynamic parameters as fluorescence versus intensity data, if the above equation is used (17). Also, it must also be realized that anisotropy measurements have more noise than simple intensity measurements, so that it is preferable to just make intensity measurements, both because of the better quality of the latter data and the more straightforward analysis. 

Photoluminescence measurements are inherently more sensitive than absorption, enabling detection limits of 10 mol dm to be readily achieved. Luminescence intensity and lifetime are the most commonly monitored properties however fluorescence anisotropy, spectral shifts, and changes in vibrational fine-structure may all be used as probing parameters. 

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