Fluorescent anisotropy measurements

In practice, fluorescence anisotropy measurements are carried out as described by Larsson et al. [134] using ... 

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. 

FIGURE 5.20 (See color insert following page 280.) Temperature influence for fluorescence anisotropy measurements on C22-modified sihca. (a) Decay cnrves for DPH at different temperatures, (b) schematic of wobble motion of DPH and resulting finorescence lifetimes (xp) and half-cone angles ). (Reprodnced from Pursch, M., et al., J. Am. Chem. Soc., 121, 3201, 1999. With permission.)... 

Theoretical and experimental aspects of time-resolved fluorescence anisotropy measurements have been described in detail in the literature.174" ... 

The time and wavelength resolved fluorescence dynamics of bianthryl has been investigated by several groups [30, 82, 132, 133, 115, 116]. In addition, this molecule has been studied by picosecond absorption spectroscopy [115], electric field induced fluorescence anisotropy measurements [117] and optically induced dielectric absorption (microwave) measurements [118, 119]. The results are generally in accord with the theoretical model presented in Sections III.A and III.B. One of the challenges of studying the photodynamics of BA is that the LE and CT interconversion is so rapid (i.e., on the time scale of solvation) that it is necessary to employ ultraviolet subpicosecond and even femtosecond fluorescence spectroscopy which has only recently become available [30, 82, 132, 133]. 

Carvedilol (11), which contains a CBZ moiety, is a multiple-action antihypertensive drug that has been shown to protect cell membranes from lipid perox-idative damages. Cheng et al. have studied [131] carvedilol, CBZ and 4-hydroxy-carbazole in a 9 1 DMPC DMPG membrane. Fluorescence anisotropy measurement showed that carvedilol is relatively mobile and does not have a rigidly defined molecular orientation in the membrane. The fluorescence spectra... 

Dynamic fluorescence anisotropy is based on rotational reorientation of the excited dipole of a probe molecule, and its correlation time(s) should depend on local environments around the molecule. For a dye molecule in an isotropic medium, three-dimensional rotational reorientation of the excited dipole takes place freely [10]. At a water/oil interface, on the other hand, the out-of-plane motion of a probe molecule should be frozen when the dye is adsorbed on a sharp water/oil interface (i.e., two-dimensional in respect to the molecular size of a probe), while such a motion will be allowed for a relatively thick water/oil interface (i.e., three-dimensional) [11,12]. Thus, by observing rotational freedom of a dye molecule (i.e., excited dipole), one can discuss the thickness of a water/oil interface the correlation time(s) provides information about the chemi-cal/physical characteristics of the interface, including the dynamical behavioiu of the interfacial structure. Dynamic fluorescence anisotropy measurements are thus expected... 

The laboratory coordinate system chosen for TIR fluorescence anisotropy measurements is illustrated in Figure 12.2. SRIOI molecules located at a water/oil interface (in the x-y plane) are excited by an s-polarized laser beam along the x -axis. The TIR fluorescence is then detected along the z-axis and its polarization is selected by a polarizer. The fluorescence decay profile observed under such a configuration is analysed for two limiting cases, depending on the structure of a water/oil interface two-dimensional or three-dimensional. 

In this case, r(0) and the magic angle are calculated to be 0.4 and 54.7°, respectively. The thickness of a water/oil interfacial layer would be evaluated through TIR fluorescence anisotropy measurements and the value(s) provides information about characteristic features at a water/oil interface. 

TABLE 12.3. Magic angles for the TIR fluorescence anisotropy measurements and structural dimensions at the interfaces. 

Bene B, Fulwyler MI, Damjanovich S. Detection of receptor clustering by flow cytometric fluorescence anisotropy measurements. Cytometry 2000 40 292-306. 

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. 

A treatment for analysing the excitation and fluorescence multiwavelength polarized decay surfaces has been given for the case of a mixture of noninteracting species. An improved model for analysis of fluorescence anisotropy measurements has been presented. Limitations to the use of intense excitation pulses in fluorescence and thermal lens spectrophotometers are discussed in terms of optical saturation. Such artefacts can be eliminated by reference to the fluorescence quantum yield of Rhodamine 6G. A model has been given to describe spectral diffusion in time-resolved hole-burning spectroscopy. ... 

Time-resolved fluorescence spectroscopy and fluorescence anisotropy measurements have been applied to study (i) excimer formation and energy transfer in solutions of poly(acenaphthalene) (PACE) and poly(2-naphthyl methacrylate) (P2NMA) and (ii) the conformational dynamics of poly(methacrylic acid) (PMA) and poly (acrylic acid) as a function of solution pH. For PACE and P2NMA, analysis of projections in which the spectral, temporal and intensity information are simultaneously displayed have been used to re-examine kinetic models proposed to account for the complex fluorescence decay behaviour that is observed. Time-resolved fluorescence anisotropy measuranents of fluorescent probes incorporated in PMA have led to the proposal of a "connected cluster" model for the hypercoiled conformation of this polymer existing at low pH. 

A further application of time-resolved fluorescence measurements is in the study of conformational dynamics of polymer chains in solution. Fluorescence anisotropy measurements of macromolecules incorporating suitable fluorescent probes can give details of chain mobility and polymer conformation (2,14). A particular example studied in this laboratory is the conformational changes which occur in aqueous solutions of polyelectrolytes as the solution pH is varied (15,16). Poly(methacrylic acid) (PMA) is known to exist in a compact hypercoiled conformation at low pH but undergoes a transition to a more extended conformation at a degree of neutralization (a) of 0.2 to 0.3 (1 6). Similar conformational transitions are known to occur in biopolymer systems and consequently there is considerable interest in understanding the nature of the structures present in model synthetic polyelectrolyte solutions. 

An Applied Photophysics Model SP 2X nanosecond spectrometer incorporating an alternating polarization rotation unit ( ) was used for the time-resolved fluorescence anisotropy measurements. An excitation wavelength of 365 nm was employed for excitation of the anthracene end-groups and emission above 400 nm was isolated with a Schott GG 400 filter. 

In particular, the application of multi-exponential decay kinetics anticipated from models that assume distinct photophysical species within polymer chains may be inappropriate in some cases. The possibility of non-exponential fluorescence decay behaviour arising from energy migration and trapping (11) should also be considered. Additional studies of the mobilities of fluorescent probes incorporated in PMA using time-resolved fluorescence anisotropy measurements provide further support for a "connected cluster" model to describe the conformation of this polyelectrolyte in aqueous solution at low pH. 

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