Fluorescence depolarization measurements

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.

Fig. 6. g-micelle volumes ( aggregation numbers = acid residues per aggregate) in ml as determined by fluorescence depolarization measurements versus sulfonate concentration in g-equiva-lents per liter of dinonylnaphthalene sulfonates at 25 °C in benzene saturated with water [J. Colloid Sci. 12, 465 (1957)]... 

Micelle volumes estimated from fluorescence depolarization measurements have been found to agree well with those computed from osmotic pressure determinations... 

Thus, for fluorescence depolarization measurements in which the second order correlation furKtion is desired... 

The flexibility was more restricted in the nurse shark IgM. The above results, together with a steady-state fluorescence depolarization study on the flexibility of immunoglobulins from amphibia and reptiles has suggested that the degree of flexibility decreases with the level of phytogeny. Thus, time-resolved fluorescence depolarization measurements may provide one of the first techniques capable of indicating... 

Steady-state depolarization measurements of the fluorescence of probe molecules dispersed in the hydrocarbon interior of a lipH bilayer have been used extensively as a probe of the lipid microviscosity. This applkation has been reviewed Two inherent assumptions in the method, however, cast some doubt on the results obtained. Firstly the probe molecule is assumed to be a rigid spherical body rotating isotropically, a situation unlikely considering the heterogeneous nature of the system. Secondly, it is amimed that the hydrocarbon interior can be modelled using homogeneous solvents. These problems can be lai ely overcome with the use of time-resolved fluorescence depolarization measurements. 

From time-resolved fluorescence depolarization measurements, the anisotropy decay times (0) and the associated anisotropy ([>) have been determined for all first generation dendrimers using Eq.(l) ... 

From fluorescence depolarization measurements, anisotropy relaxation times and the associated anisotropy values have been determined for p-C2P1 p-C2P2, p-C2P3, and / -C2P. For the dendrimers with more than one chromophore, a two-exponen-tial function was found to be necessary to fit the experimental anisotropy decay traces (Table 1.2). The multichromophoric dendrimers present two-exponential decays in the anisotropy traces. The fast component (410 ps to 280 ps) of the anisotropy decay (Table 1.2) is found to decrease from p-C2P2 to p-C2P4. Contrary to the meta-substituted dendrimers m-C 1 P , the sum of the / , is now always close to the limiting value of the anisotropy even if 11 is larger than one. 

In the current paper we extend previous work on poly(methyl acrylate) (9 - 10) and poly(methyl methacrylate) (9 - 11) in which the phosphorescence depolarization technique was shown to provide data which were consistent with reported dielectric and mechanical relaxation experiments, to the study of the molecular behaviour of poly(n-butyl methacrylate). This polymer, whilst of technological application, has received much lesser attention using conventional dynamic relaxation techniques than has been devoted to PMA and PMMA. In addition, fluorescence depolarization measurements have been employed in an attempt to provide complementary information regarding the higher frequency behaviour of the polymer. 

Fluorescence depolarization measurements of aromatic residues and other probes in proteins can provide information on the amplitudes and time scales of motions in the picosecond-to-nanosecond range. As for NMR relaxation, the parameters of interest are related to time correlation functions whose decay is determined by reorientation of certain vectors associated with the probe (i.e., vectors between nuclei for NMR relaxation and transition moment vectors for fluorescence depolarization). Because the contributions of the various types of motions to the NMR relaxation rates depend on the Fourier transform of the appropriate correlation functions, it is difficult to obtain a unique result from the measurements. As described above, most experimental estimates of the time scales and magnitudes of the motions generally depend on the particular choice of model used for their interpretation. Fluorescence depolarization, although more limited in the sense that only a few protein residues (i.e., tryptophans and tyrosines) can be studied with present techniques, has the distinct advantage that the measured quantity is directly related to the decay of the correlation function. 

Fig. 1.12 Geometry of experimental set-ups employed in fluorescence depolarization measurements.

Table 1.4 Rotational correlation times x, of proteins in aqueous solution at 25 °C determined by time-resolved fluorescence depolarization measurements [37].

We have performed fluorescence depolarization measurements on both compressed and uncompressed gels, containing similar concentrations of chlorosomes. To obtain good results the absorption in the excitation band was kept low, typically 0.05. We excited at 460 nm and detected at 750 nm. Several thousands of counts were needed to minimize statistical errors. To keep the measuring time low, bandwidths of 16 nm were used. The determination of 1 intensity took 30 seconds. To eliminate the effect of fluctuating lamp intensities, 20 series of measurements were poformed on both a compressed and an uncompressed gel. One series consists of the determination of 4 intensities for both the uncompressed and the compressed gel. From one series the parameters , , , and the constant C were determined. These led to the following average parameters and standard errors ... 

In conclusion, the method of fluorescence depolarization measurements on particles in compressed gels can be performed on chlorosomes and they lead to results, which are in good agreement with the theoretical predictions from [1], and results fromt linear dichroism measurements [2]. The results support the further use of the expressions in... 

Hydrolytic digestion of pullulan with pullulanase and subsequent release of FITC moiety from the surface of liposomes were examined by a decrease in the steady-state fluorescence depolarization. Measurements were run on a Union Giken fluorescence polarization spectrophotometer FS-501S, where the FITC fluorescence (520 nm) was detected by exciting the sample at 440 nm using a sharp cut-off filter Y46 (Hoya Glass Works, Tokyo). To a 3.0 ml solution of liposome (1.0 xio" M as egg PC) coated with a given amount of FITC(0.54)-OPP-50(1.8) was added 20 yl of pullulanase solution (2.0 unit) after preincubation for 5 min in 200 mM Tris-HCl (pH 8.0) at 25.0 °C. 

For cytochrome c oxidase, distances between subunit It, heme a and cytochrome c bound to subunit III could be estimated.Subunit II was labelled at a thiol group with A -(iodoacetamidoethyl)-l-aminonaphthalene-5-sulphonic acid and subunit III with either thionitrobenzoate-activatcd cytochrome c or a fluorescent porphyrin analogue. In this study factors allowing for the probable orientation of groups were included, being estimated from fluorescence depolarization measurements. The distances between subunit II and heme a, subunit II and cytochrome c (bound to subunit III) and between cytochrome c (bound to subunit III) and heme a were estimated to be 52, 35, and 25 A respectively. 

Limpouchova Z, Prochazka K, Fidler V, Dvorak J, Bednar B (1993) Molecular-movements and dynamics in solutions studied by fluorescence depolarization measurement. Collect Czech Chem Commun 58(2) 213-233. doi 10.1135/ccccl9930213... 

Gochanour CR, Payer MD (1981) Electronic excited-state transport in random-systems— time-resolved fluorescence depolarization measurements. J Phys Chem 85(14) 1989-1994. doi 10.1021/j150614a008... 

Fig. 44. Experimental geometty for time-iesolved fluorescence depolarization measurements...

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