Anisotropy decay traces

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 contrast to the monoexponential anisotropy trace of monochromophoric p-CIP], the corresponding traces of the multichromophoric dendrimers p-ClP3 and p-C1P4 reveal a second and fast anisotropy decay component on the order of 50-80 ps (Table 1.1). Within the framework of the Forster... 

StudiN of DNA by fluorescence can be traced to the use of dyes lo stain chromatin for fluorescence microscopy. The use of time-resolved fluorescence for DNA dynamics originated with the measurement of anisotropy decays of EB bound to DNA. " These early studies showed an unusual anisotropy decay, similar to that found for DPH in membranes, in which the anisotropy at long times did not decay to zero (Figure 11,24). At that time, the results were interpreted in terms of the angle through which the EB could rotate within the DNA helix. However, more recent... 

The theory for rotational diffusion of ellipsoids, and measurements by fluorescence polarization, can be traced to the classic reports by F. Perrin. Since these seminal reports, the theory has been modified to include a description of expected anisotropy decays. Hiis theory has been summarized in several reviews.For a rigid ellipsoid with three unequal axes, it is now agreed that the anisotropy decays with five correlation times. The correlation times depend on the three rotational diffiision coefficients, and the amplitudes depend on the orientation of the absorption and emission transition moments widiin the fluoroi iore and/or ellipsoid. While the the( predicts five correlation times, it is known diat two pairs of correlation times will be very close in magniOide, so that in practice only three correlation times are expect for a nonsf oical molecule. ... 

Johnson, et a/. (27) examine fluorescence anisotropy decay of center-labeled polyisoprene and of the small molecule probe 9,10-diphenylanthracene in poly-isoprene tetrahydrofuran solutions at several temperatures as the concentration of polyisoprene is increased from trace to the bulk. Measurements and fits appear in Figure 6.6. The transition dipoles of the center label and the small-molecule... 

Fluorescence anisotropy of N-acetyl-tryptophananide has been measured (18) in various environments. This experiment was particularly difficult because it required excitation at the fourth harmonic (266 nm) with limited energy (2yJ) and detection in the UV at 340 nm. Fig. 3 shows the result of increasing viscosity on the decay time of the anistropy calculated using Eq. l. In order to obtain these results, 400 traces of I(t) and Ij.(t) were averaged. 

Figure 47. Top Experimental fluorescence decays corresponding to the excitation and detection of the S, - Og band of jet-cooled r-stilbene expansion orifice 70 fim, 75 psig Ne backing pressure, nozzle T 150°C, laser-to-nozzle distance 3 mm. Bottom Fluorescence anisotropies r(t). The experimental trace was obtained directly from the parallel and perpendicular decays at the top of the figure using the expression for r(t). The upper theoretical trace was obtained from decays calculated for an asymmetric top (rotational constants 2.678,0.262, and 0.250 GHz) at 5 K with convolution of the experimental response function accounted for. The bottom trace was calculated from the bottom two decays (symmetric top) of Fig. 46.

Figure 48. Comparison of experimental and simulated fluorescence polarization anisotropies for the S, + 789 cm 1 excitation of jet-cooled r-stilbene. The anisotropies include convolution effects associated with the finite excitation pulse width. The upper trace was calculated using the theoretical results of Ref. 49. The lower trace was obtained from experiment. The inset shows a simulated decay for this excitation band. 

Figure 5.17. Time-correlated single photon couming traces acquired under equilibrium folding conditions with 10 pM aTS and 10 / M ANS. The vertical and horizontal polarization components of the ANS excited-state decity are shown at 0 M (a, vert cal b, hor zontal) and 3 M urea (c, vertical d. horizontal) upon excitation with 370 nm vertically polarized light. 1 he fast component corresponds to the decay of unbound ANS. The corresponding calculated anisotropy curves at 0 and 3 M (solid and dotted trace, respectively] are also shown (inset). The smooth lines represent fits to the associative model Source Bilsel. O., Yang. L. Zitzewitz, J. A, Beechem. J. M. and Mattliews, C. R. 1999, Biochemistry. 38.4177 — 4187. Authorization of reprint accorded by the American Chemical Society.

FA decay can be acquired using a TCSPC equipped with two polarizers for the excitation and emission respectively. Also in this case two signals are measured setting the polarizer first parallel and then perpendicular. The decay of the anisotropy is calculated from the two intensity decays. If an L geometry is used and the two traces are measured sequentially it is important to use the same experimental conditions and in particular the same acquisition time for both measurements. 

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