Fluorescence anisotropy ratio

In a practical experiment, a correction factor is introduced to take into account the sensitivity of the fluorescence spectrometer to anisotropy. The factor G = /hv//hh is used, where the subscript v and h correspond to the vertically polarized light (z direction) and the horizontally polarized light (x- and -axes), respectively, with the incident light polarized in the.v direction. Taking this correction factor into account, the value of the fluorescence anisotropy ratio is... 

Fig. 16. Fluorescence and excitation spectra and the fluorescence anisotropy ratio as a function of wavelength within the fluorescence and excitation bands for CHA-s-BPDA-CHA molecularly dispersed in PMMA (c = 10 M).

Characterization of the polarization state of fluorescence (polarization ratio, emission anisotropy)... 

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. 

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)... 

Equation (11.9) indicates the possibility of calculating the rotational correlation time of the fluorophore not only by varying the T/q ratio but also by adding a collisional quencher. Interaction between the quencher and fluorophore decreases the fluorescence lifetime and intensity of the fluorophore, and increases its fluorescence anisotropy. Plotting 1 /A as a function of r0 yields a straight line with a slope equal to r. If the fluorophore is tightly bound to the macromolecule and does not exhibit any residual motions, the measured 0r is equal to, and the extrapolated anisotropy is equal to that measured at a low temperature. 

Figure 11.4 Steady-state fluorescence anisotropy vs. temperature/viscosity ratio for tryptophan residues of cytochrome b2 core. Data are obtained by thermal variations in the range 10-36°C.

Fig. 7.12. Steady-state fluorescence anisotropy as a function of the Zn +/monomer ratio. (Reprinted with permission from ref. [28]. Copyright 2005 American Chemical Society).

Fluorescence depolarization time courses contain information about rotational diffusion (26). The main difficulty with streak camera measurements of time-resolved fluorescence depolarization is limited signal-to-noise ratio of single shot records. The fluorescence anisotropy parameter is defined as s... 

Figure 8.21. Steady-state fluorescence anisotropy vs. temperature over viscosity for 5 pM Calcofluor in the presence of 10 pM sialylated ai-acid glycoprotein (A ex, 300 nm Xem, 435 nm) (plot a), and for 8.5 pM Calcofluor in the presence of 5.5 pM asialylated Uj- acid glycoprotein ((A ex, 300 nm, A em, 445 nm (plot b). The data shown are the mean values of two measurements, and they are obtained by thermal variation in the range 15-35°C. The ratio T/p is expressed in Kelvins/centipoise. Source Albani, J. R., Sillen A., Coddeville, B., Plancke, Y. D., and Engelborghs, Y. 1999, Carbohydr. Res. 322, 87-94.

Semi-rigid s-BPDA-PDA also shows similar effect of T[ or final cure temperature, Tf, on the morphology [74,81-83]. A sign of crystallization is observed at T[ or Tf = 400°C. Unfortunately, there are so far no reports on the S AXS measurements for the s-BPDA-PDA film. An increase in Tf also led to a decrease in the anisotropy ratio, r, for the CT fluorescence concurrently with the increases in film density and CT fluorescence intensity. This result can be rationalized in terms of an increased efficiency of the non-radiative energy migration between the CTCs, caused by the annealing-induced CTC population increase [49]. 

The sensitivity, dynamic range, selectivity and stability are the key factors that determine the sensor performance. The current PEBBLE technologies have relied on fluorescence emission ratios for signal transduction (though fluorescence anisotropy and frequency modulation were also tested) and the performance of the PEBBLEs was established using either an Olympus IMT-II (Lake Success, NY, USA) inverted fluorescence microscope or a Fluoro-Max 2 spectrofluorometer (ISA Jobin Yvon-Spex, Edison, NJ, USA). The spectra and confocal images for the intracellular measurements were acquired with the same inverted fluorescent microscope and a Perkin Elmer UltraView confocal microscope system equipped with an Ar-Kr laser. 

The emission spectra of the unbound TPPS display a two-banded feature with a main higher energy peak at 642 nm. In the case of a 1 1 molar ratio, this component is located at about 642-643 nm and shows very low fluorescence anisotropy. At higher CD concentration, the maxima move to 650 nm, displaying a larger anisotropy. 

Fig. 5. Time-resolved fluorescence anisotropy [r t)] of TPPS/CD 3 at different molar ratios.

Anisotropy describes the rotational dynamics of reporter molecules or of any sensor segments to which the reporter is rigidly fixed. In the simplest case when both the rotation and the fluorescence decay can be represented by single-exponential functions, the range of variation of anisotropy (r) is determined by variation of the ratio of fluorescence lifetime (xF) and rotational correlation time ([Pg.9]

In the expression of the polarization ratio, the denominator represents the fluorescence intensity in the direction of observation, whereas in the formula giving the emission anisotropy, the denominator represents the total fluorescence intensity. In a few situations (e.g. the study of radiative transfer) the polarization ratio is to be preferred, but in most cases, the use of emission anisotropy leads to simpler relations (see below). 

---