Fluorescence, depolarization

Fluorescence spectroscopy is discussed in Chapter 17. Here we describe the experimental method for determining the rotational diffusion coefficient. The parameter we are interested in is pjj, which is the harmonic mean of the two 

Here P is the degree of polarization of fluorescent light emitted at right angles to the direction of the incident light, tq is the lifetime of the excited state of the fluorescence, and Pq is an empirical constant. The rotational diffusion coefficient 0h is obtained from 

Mathematics of Diffusion, 2nd ed. Oxford Oxford University Press, 1975. Einstein, A., Ann. Phys. 17, 549 (1905). 

Fourier, J. B. J., Theorie Analytique de la Chaleur (Paris, 1822), translated by A. Freeman, Cambridge, England, 1878, and New York, 1955. (See Grattan-Gnirmess, I., Joseph Fourier 1768-1830. Cambridge, MA MIT Press, 1972.) 

1 Show that Pick s second law (a) can be written in the form 

Plotting 1 / P as a function of T / r yields in principle a linear graph with a y-intercept equal to 1 / Po and a slope equal to 

Decreasing the temperature or increasing the concentration of sucrose and / or glycerol in the medium, increases the viscosity of the medium. 

Whether the fluorophores are intrinsic or extrinsic to the macromolecule (protein, peptide or DNA), depolarization is the result of two motions, the local motions of the fluorophore and the global rotation of the macromolecule. 

The rotational correlation time of a spherical macromolecule can be determined with one of equations 5.15 and 5.16  

Equations 5.15 and 5.16 do not yield exactly the same value for ( )p For example, for a protein with a molecular mass of 235 kDa such as flavocytochrome ba extracted from the yeast Hansenula anomala, the rotational correlation times calculated from Eq. 5.15 and 5.16 are 97 and 90 ns, respectively. 

In vitrified solution and/or when fluorophore molecules do not show any residual motions, the measured polarization is equal to the intrinsic one, P0. This value is obtained at low 

However, during the excited-state lifetime, energy transfer to neighboring molecules and/or local and global motions of the fluorophores can be observed. These two phenomena induce reorientation of the emission dipole, thereby depolarizing the system. Therefore, the value of the measured polarization P will be lower than intrinsic polarization value PQ (Weber, 1952). 

---

Micellar structure has been a subject of much discussion [104]. Early proposals for spherical [159] and lamellar [160] micelles may both have merit. A schematic of a spherical micelle and a unilamellar vesicle is shown in Fig. Xni-11. In addition to the most common spherical micelles, scattering and microscopy experiments have shown the existence of rodlike [161, 162], disklike [163], threadlike [132] and even quadmple-helix [164] structures. Lattice models (see Fig. XIII-12) by Leermakers and Scheutjens have confirmed and characterized the properties of spherical and membrane like micelles [165]. Similar analyses exist for micelles formed by diblock copolymers in a selective solvent [166]. Other shapes proposed include ellipsoidal [167] and a sphere-to-cylinder transition [168]. Fluorescence depolarization and NMR studies both point to a rather fluid micellar core consistent with the disorder implied by Fig. Xm-12. 

Tao T 1969 Time-dependent fluorescence depolarization and Brownian rotational diffusion coefficients of macromolecules Biopolymers 8 609-32... 

Other solubilization and partitioning phenomena are important, both within the context of microemulsions and in the absence of added immiscible solvent. In regular micellar solutions, micelles promote the solubility of many compounds otherwise insoluble in water. The amount of chemical component solubilized in a micellar solution will, typically, be much smaller than can be accommodated in microemulsion fonnation, such as when only a few molecules per micelle are solubilized. Such limited solubilization is nevertheless quite useful. The incoriDoration of minor quantities of pyrene and related optical probes into micelles are a key to the use of fluorescence depolarization in quantifying micellar aggregation numbers and micellar microviscosities [48]. Micellar solubilization makes it possible to measure acid-base or electrochemical properties of compounds otherwise insoluble in aqueous solution. Micellar solubilization facilitates micellar catalysis (see section C2.3.10) and emulsion polymerization (see section C2.3.12). On the other hand, there are untoward effects of micellar solubilization in practical applications of surfactants. Wlren one has a multiphase... 

Characterization of Aquatic Humic Acid Fractions by Fluorescence Depolarization Spectroscopy... 

This chapter presents new information about the physical properties of humic acid fractions from the Okefenokee Swamp, Georgia. Specialized techniques of fluorescence depolarization spectroscopy and phase-shift fluorometry allow the nondestructive determination of molar volume and shape in aqueous solutions. The techniques also provide sufficient data to make a reliable estimate of the number of different fluorophores in the molecule their respective excitation and emission spectra, and their phase-resolved emission spectra. These measurements are possible even in instances where two fluorophores have nearly identical emission specta. The general theoretical background of each method is presented first, followed by the specific results of our measurements. Parts of the theoretical treatment of depolarization and phase-shift fluorometry given here are more fully expanded upon in (5,9-ll). Recent work and reviews of these techniques are given by Warner and McGown (72). 

Steady-State Fluorescence Depolarization Spectroscopy. For steady state depolarization measurements, the sample is excited with linearly polarized lig t of constant intensity. Observed values of P depend on the angle between the absorption and emission dipole moment vectors. In equation 2 (9), Po is the limiting value of polarization for a dilute solution of fluorophores randomly oriented in a rigid medium that permits no rotation and no energy transfer to other fluorophores ... 

Time Resolved Fluorescence Depolarization. In Equation 3, it is assumed that the polarization decays to zero as a single exponential function, which is equivalent to assuming that the molecular shape is spherical with isotropic rotational motion. Multiexponential decays arise from anisotropic rotational motion, which might indicate a nonspherical molecule, a molecule rotating in a nonuniform environment, a fluorophore bound to tbe molecule in a manner that binders its motion, or a mixture of fluorophores with different rotational rates. 

The fluorescence depolarization technique excites a fluorescent dye by linearly polarized light and measures the polarization anisotropy of the fluorescence emission. The fluorescence anisotropy, r, is defined as... 

Figure 4.6 shows an apparatus for the fluorescence depolarization measurement. The linearly polarized excitation pulse from a mode-locked Ti-Sapphire laser illuminated a polymer brush sample through a microscope objective. The fluorescence from a specimen was collected by the same objective and input to a polarizing beam splitter to detect 7 and I by photomultipliers (PMTs). The photon signal from the PMT was fed to a time-correlated single photon counting electronics to obtain the time profiles of 7 and I simultaneously. The experimental data of the fluorescence anisotropy was fitted to a double exponential function. 

Figure 4.6 Block diagram of the apparatus for the fluorescence depolarization measurement. The dashed and solid arrows indicate the light paths ofthe excitation pulse and the fluorescence from the sample. OBJ microscope objective, M mirror, L lens, DM dichroic mirror, LP long-pass filter, PH pin-hole, PBS polarizing beam splitter, P polarizer, PMT photomultiplier.

Aoki, H., Kitamura, M. and Ito, S. (2008) Nanosecond dynamics of poly(methyl methacrylate) brushes in solvents studied by fluorescence depolarization method. Macromolecules, 41, 285-287. 

Horinaka, J., Ono, K. and Yamamoto, M. (1995) Local chain dynamics of syndiotactic poly(methyl methacrylate) studied by the fluorescence depolarization method. Polym. J., 27, 429-435. 

Unilamellar vesicles (PC, DPPC) Polar head/acyl core Fluorescence depolarization (DSHA) 32 450... 

The present experiments are mute as to the timescale on which delocalization may occur. EPR results on Ru(bpy)"5 demonstrate localization of the bpy electron density in this Ru(II)(bpy)2 (bpy )+ species on the EPR timescale, but suggest that delocalization may occur on a timescale only slightly longer. It is possible that either time-resolved EPR or temperature dependent fluorescence depolarization experiments may establish the time-scale of localization in Ru(bpy) +. 

Fluorescence depolarization of 9-anthryloxy-fatty acids in CTABr... 

C.H. Lochmiiller and S.S. Saavedra, Conformational changes in a soil fulvic acid measured by time-dependent fluorescence depolarization, Anal. Chem., 58... 

F. Perrin Discussion on Jean Perriris diagram for the explanation of the delayed fluorescence by the intermediate passage through a metastable state First qualitative theory of fluorescence depolarization by resonance energy transfer... 

If excited molecules can rotate during the excited-state lifetime, the emitted fluorescence is partially (or totally) depolarized (Figure 5.9). The preferred orientation of emitting molecules resulting from photoselection at time zero is indeed gradually affected as a function of time by the rotational Brownian motions. From the extent of fluorescence depolarization, we can obtain information on the molecular motions, which depend on the size and the shape of molecules, and on the fluidity of their microenvironment. 

Lipari G. and Szabo A. (1980) Effect of Vibrational Motion on Fluorescence Depolarization and Nuclear Magnetic Resonance Relaxation in Macromolecules and Membranes, Biophys. J. 30, 489—506. Steiner R. F. (1991) Fluorescence Anisotropy Theory and Applications, in Lakowicz J. R. (Ed.), Topics in Fluorescence Spectroscopy, Vol. 2, Principles, Plenum Press, New York, pp. 127-176. 

Lipari G. and Szabo A. (1980) Effect of Vibrational Motion on Fluorescence Depolarization and Nuclear Magnetic Resonance Relaxation in Macromolecules and Membranes, Biophys. J. 30, 489-506. 

In the authors laboratory we have studied the fluorescence depolarization of IR-140 in lipid bilayer membranes of L-a-dipalmitoylphosphatidylcholine (DPPC) and observed similar differences between the gel and liquid crystalline phases as has been widely reported for UV/visible probes such as l,6-diphenyl-l,3,5-hexatriene (DPH) in this same medium. Figure 12.4 shows some of these results. 

A. Kasprzak and G. Weber, Fluorescence depolarization and rotational modes of tyrosine in bovine pancreatic trypsin inhibitor, Biochemistry 21, 5924-5927 (1982). 

J. R. Lakowicz and B. Maliwal, Oxygen quenching and fluorescence depolarization of tyrosine residues in proteins, J. Biol. Chem. 258, 4794-4801 (1983). 

Analysis of rotational mobility of fluorophores by observation of fluorescence depolarization with nanosecond time resolution(28) or by variation of the lifetime (by the action of quenchers ).(9,29 30)... 

Figure 2.4. Schematic representation of processes which lead to fluorescence depolarization in proteins rotation of the protein molecule as a whole with correlation time rotation of the fluorophore with correlation time d, and excitation energy transfer, represented by the wavy arrow.

T. Ichiye and M. Karplus, Fluorescence depolarization of tryptophan residues in proteins A molecular dynamics study, Biochemistry 22, 2884-2894 (1983). 

Fluorescence depolarization by excitation transfer between intercalated ethidium dyes was originally studied in attempts to determine their unwinding angle/65, 156l5S i The total anisotropy was assumed to be given by a simple... 

F. W. J. Teale, Fluorescence depolarization by light scattering in turbid solutions,... 

L. A. Chen, R. E. Dale, S. Roth, and L. Brand, Nanosecond time-dependent fluorescence depolarization of diphenylhexatriene in dimyristoyllecithin vesicles and the determination of microviscosity, J. Biol. Chem. 252, 2163-2169 (1977). 

---