Time resolved fluorescence anisotropy

Determination of fluorescence anisotropy decay involves the acquisition of fluorescence decays using distinct combinations of excitation and emission polarizers, in accordance with Equation (12.6)  

The discrimination of the individual correlation times, ) and their assignment to the fluorophores, fluorophore aggregates or fluorophore-matrix assemblies can be obtained by solving Equation (12.1) and the following equations (Equations 12.7 and 12.8) [3, 7]  

In the previous paragraphs we have discussed the basic aspects of steady state fluorescence anisotropy. It is, on the other hand possible to investigate the time dependency of the FA anisotropy of a system excited with a pulsed source [46] This technique allows to study the rate of the operating depolarization processes and becomes very useful especially in those cases in which excited states deactivation is much slower than depolarization. In such a situation, in fact, most of the light emitted is not polarized and steady state techniques give poor information. 

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Cross A J and Fleming G R 1984 Analysis of time-resolved fluorescence anisotropy decays Blophys. J. 46 45-56... 

Matro A and Cina J A 1995 Theoretical study of time-resolved fluorescence anisotropy from coupled chromophore pairs J. Phys. Chem. 99 2568-82... 

Nakano, M., Kamo, T., Sugita, A. and Handa, T. (2005) Detection of bilayer packing stress and its release in lamellar-cubic phase transition by time-resolved fluorescence anisotropy. Journal of Physical Chemistry B, 109 (10), 4754—4760. 

Fig. 4.9. Schematic of time-resolved fluorescence anisotropy sample is excited with linearly polarized light and time-resolved fluorescence images are acquired with polarization analyzed parallel and perpendicular to excitation polarization. Assuming a spherical fluorophore, the temporal decay of the fluorescence anisotropy, r(t), can be fitted to an exponential decay model from which the rotational correlation time, 6, can be calculated.

Siegel, J., Suhling, K., Leveque-Fort, S., Webb, S. E. D., Davis, D. M., Phillips, D., Sabharwal, Y. and French, P. M. W. (2003). Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) Imaging the rotational mobility of a fluorophore. Rev. Sci. Instrum. 74, 182-92. 

Time-resolved emission anisotropy experiments provide information not only on the fluidity via the correlation time rc, but also on the order of the medium via the ratio rco/ro. The theoretical aspects are presented in Section 5.5.2, with special attention to the wobble-in-cone model (Kinosita et al., 1977 Lipari and Szabo, 1980). Phospholipid vesicles and natural membranes have been extensively studied by time-resolved fluorescence anisotropy. An illustration is given in Box 8.3. 

Berberan-Santos M. N., Choppinet P., Fedorov A., Jullien L., Valeur B. (1999) Multichromophoric Cydodextrins. 6. Investigation of Exdtation Energy Hopping by Monte-Carlo Simulations and Time-Resolved Fluorescence Anisotropy, J. Am. Chem. Soc. 121, 2526-2533. 

In a totally independent approach, highly fluorescent but rigid molecules can be used and their reorientational movement probed in solution, mostly by time-resolved fluorescence anisotropy, (85) For rodlike molecules likep-oligo-phenyls, this movement corresponds to the reorientation of the long molecular axis the length of which can be easily varied. 86 The use of fluorescence probes in liquid and solid media and on surfaces has been treated recently in several reviews and books. 87 ... 

D. J. S. Birch, A. S. Holmes, J. R. Gilchrist, R. E. Imhof, S. M. A1 Alawi and B. Nadolski, A multiplexed single-photon instrument for routine measurement of time-resolved fluorescence anisotropy, J. Phys. E Sci. Instrum. 20, 471-473 (1987). 

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. 

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)... 

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)... 

M. Vincent and J. Gallay, Time-resolved fluorescence anisotropy study of effect of a cis double bond on structure of lecithin and cholesterol-lecithin bilayers using n-(9-anthroyloxy) fatty acids as probes, Biochemistry 23, 6514-6522 (1984). 

J. R. Lakowicz, H. Cherek, and B. P. Maliwal, Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry, Biochemistry 24, 376-383 (1985). 

M. Vincent, J. Gallay, J. de Bony, and J.-F. Tocanne, Steady-state and time-resolved fluorescence anisotropy study of phospholipid molecular motion in the gel phase using l-palmitoyl-2-[9-(2-anthryl)-nonanoyl]-sn-glycero-3-phosphocholine as probe, Ear, J. Biochem. 250, 341-347 (1985). 

H. Fukumura and K. Hayashi, Time-resolved fluorescence anisotropy of labeled plasma proteins adsorbed to polymer surfaces, J. Colloid Interface Sci. 135, 435M42 (1990). 

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

The ultrafast excitation-energy transfer of the J-aggregates on the octahedral AgBr is studied by the time-resolved fluorescence-anisotropy decay (r(t)) measurements [9]. They are biphasic with two time constants of -0.15 ps and 2-7 ps as shown in Fig. 6. Each phase should reflect some difference in the orientation of the dye molecules of the J-aggregates. 

Fig. 6. Time-resolved fluorescence anisotropy of Dye 1 on octahedral AgBr crystals. Smooth solid line shows the fitting curve.

A. van Hoek, and D. N. Reinhoudt, Flexibility of enzymes suspended in organic solvents probed by time-resolved fluorescence anisotropy. Evidence that enzyme activity and enantioselectidty are directly related to enzyme flexibility,... 

Munaka, T., Kanai, M., Abe, H., Fujiyama, Y., Sakamoto, T., Mahara, A., Yamay-oshi, A., Nakanishi, H., Shoji, S., Murakami, A., In situ cell monitoring on a microchip using time-resolved fluorescence anisotropy analysis. Micro Total Analysis Systems 2003, Proceedings 7th pTAS Symposium, Squaw Valley, CA, Oct. 5-9, 2003, 283-286. 

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. 

Tleugabulova, D. Czardybon, W. Brennan, J. D., Time-resolved fluorescence anisotropy in assessing side-chain and segmental motions in polyamines entrapped in sol-gel derived silica. Journal of Physical Chemistry B 2004, 108(30), 10692-10699... 

The technique of transient grating spectroscopy has been reviewed, with particular emphasis on its application to monitoring non-radiative deactivation. A unified theory of time-resolved fluorescence anisotropy and Stokes shift spectroscopy has appeared. A separate review has considered the chemical and photophysical events occurring from upper excited states as accessed by multiphoton absorption techniques. ... 

Broos J, Bisser AJW, Engbersen JI, Verboom W, van Hoek A, and Reinhoudt DN. Flexibility of Enzymes Suspended in Organic Solvents Probed by Time-Resolved Fluorescence Anisotropy. Evidence that Enzyme Activity and Enan-tioselectivity are Directly Related to Enzyme Flexibility. / Am Chem Soc 1995 117 12657-12663. 

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. 

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. 

It should be noted that the short excited-state lifetime of the 9,10-DMA probe makes the accurate determination of slower polymer rotations difficult. Preliminary studies using pyrene (a longer-lived fluorescent probe) solubilized in PMA at a =0, have indicated an additional larger value of X(. than is observed using the 9,10-DMA end-group probe (Ghiggino and Tan unpublished observations), suggesting that the polymer chain conformation may consist of clusters of various sizes. These results demonstrate the usefulness of time-resolved fluorescence anisotropy measuranents for... 

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. 

This explanation for the origin of the non-exponential decays of tryptophan fluorescence, however, cannot rationalize the results of time-resolved fluorescence anisotropy in these proteins. Beec-hem and Brand review the time-resolved fluorescence of tryptophans in proteins (32). The following conclusions concerning time-resolved fluorescence can be derived i) fluorescence decays with at least two-lifetime components, when internal rotation of tryptophan is observed from the time-resolved fluorescence anisotropy, ii) fluorescence decays... 

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