Depolarization, fluorescence polarization

To perform structural research on a food stuff into which a colorant is incorporated, special properties of fluorescing molecules are exploited fluorescence efficiency, fluorescence lifetime, fluorescence quenching, radiationless energy (Foerster) transfer, stationary or time-dependent fluorescence polarization and depolarization." Generally, if food colorants fluoresce, they allow very sensitive investigations which in most cases cannot be surpassed by other methods. 

Figure 4.1. Time scales for rotational motions of long DNAs that contribute to the relaxation of the optical anisotropy r(t). Experimental methods used to study these motions in different time ranges are also indicated along with the authors and dates of some early work in each case. FPA, Fluorescence polarization anisotropy (Refs. 15, 18-20, and 87) TPD, transient photodichroism (Refs. 28 and 62) TEB, transient electric birefringence (Refs. 26 and 27) DDLS, depolarized dynamic light scattering (Ref. 116) TED, transient electric dichroism (Refs. 25, 115, and 130) Microscopy, time-resolved fluorescent microscopy (Ref. 176).

Thomas, J. C., Allison, S. A., Appellof, C. J., and Schurr, J. M. (1980). Torsion dynamics and depolarization of fluorescence of linear macromolecules. II. Fluorescence polarization anisotropy measurements of a clean viral phi 29 DNA. Biophys. Chem. 12, 177-188. 

The fluorescence depolarization technique for mobility and ordering is based on the fact that the probability of absorption and emission is directional. Light polarized along a certain axis will preferably excite molecules oriented with their transition dipole moment in the same direction. The probability varies with cos 0, where 0 is the angle between the transition dipole moment and the electric field vector of the light. Emission of a photon obeys the same cos 0 (28) rule. That means that a molecule oriented with its transition dipole moment along the Z-axis will be likely to emit a photon with the same polarization. In the depolarization technique, polarizers are used to quantify the intensity of the parallel (ly) and perpendicular (Ij.) components to the original direction of polarization. 

The rotational reorientation times of the sample in several solvents at room temperature were measured by picosecond time-resolved fluorescence and absorption depolarization spectroscopy. Details of our experimental setups were described elsewhere. For the time-correlated single photon counting measurement of which the response time is a ut 40 ps, the sample solution was excited with a second harmonics of a femtosecond Ti sapphire laser (370 nm) and the fluorescence polarized parallel and perpendicular to the direction of the excitation pulse polarization as well as the magic angle one were monitored. The second harmonics of the rhodamine-640 dye laser (313 nm 10 ps FWHM) was used to raesisure the polarized transient absorption spectra. The synthesis of the sample is given elsewhere. All the solvents of spectro-grade were used without further purification. 

The fluorescence polarization excitation spectrum has been measured for thymine in aqueous solution. " The depolarization at the red edge is attributed to the hidden n, ir transition. Ionization of the lowest excited singlet and triplet states have been determined by the effect of pH on the absorption, fluorescence, and phosphorescence spectra of purines and pyrimidines. " Spectral, polarization, and quantum yield studies of cytidylyl-(3, 5 )-adenosine have also been published. Intermediates in the room-temperature flash photolysis of adenine and some of its derivatives have been identified hydrated electron, radical cations and anions, and neutral radicals resulting from their reactions have been assigned. Photoionization occurs via the triplet state. FMN encapsulated in surfactant-entrapped water pools interacts with polar head groups, entrapped water molecules, and outer apolar solvent. ... 

Fluorescence polarization cannot attain the +1 theoretical limits for maximum beam polarization owing to the nature of the absorption and emission processes, which usually correspond to electric dipole transitions. Although the excitation with linearly polarized radiation favours certain transition dipole orientations (hence certain fluorophore orientations, and the so-called photoselection process occurs), a fairly broad angular distribution is still obtained, the same happening afterwards with the angular distribution of the radiation of an electric dipole. The result being that, in the absence of fluorophore rotation and other depolarization processes, the polarization obeys the Lev shin-Perrin equation,... 

Molecules undergo significant rotation during the excited-state lifetime when p is much smaller than x. In this case, the fluorescence is depolarized. In the converse case, when p is larger than x, the emission is highly polarized. This means that one can think of fluorescence polarization as a measurement of the rotation speed (p) in units of the internal molecular clock (x). 

The fluorescence polarization has been observed for the FM and FE bands of pyrene adsorbed on silica gel (9). The excimer fluorescence of pyrene is completely depolarized even in viscous solvents. Rotational diffusion is involved in the process of excimer formation in solutions. The degree of polarization P for the FE fluorescence of adsorbed pyrene has non-zero positive... 

The depolarization of fluorescence has been observed at temperatures around 100 K not only for 1, but also for the two derivatives, lb and Ic. in contrast, the measurements performed under the same conditions for le revealed no sign of depolarization. The textbook values of the anisotropy were obtained, i.e. 0.4 and about -0.2 for excitation into Sj and S2, respectively (Fig. 8.11). The octaethyl derivative le is the porphycene with the largest separation (2.80 A) between the hydrogen-bonded nitrogen atoms and should therefore exhibit the slowest tautomerization kinetics. It was thus concluded that the reduced anisotropy values observed in three different porphycenes are caused by excited state tautomerization [30, 80]. As shown in Fig. 8.12, the interconversion between the two trans tautomers changes the direction of the transition moment. Therefore, only a part of the excited state population emits fluorescence polarized parallel to that of the transition moment in Sg-Sj absorption (this fraction should approach 0.5 if the tautomerization is fast compared to the excited state lifetime). For the remaining frac-... 

The temperature-dependent fluorescence polarization data for HP in DPPC liposomes loaded with increasing concentrations of Choi have an interesting interpretation. At high concentrations, HP was distributed close to the inner polar headgroups, which was confirmed by a low critical value of phase transition for DPPC at 31°C rather than the typical temperature at T = 41°C. However, when 20% Choi was added a depolarization effect was observed because HP was at the aqueous interface and was not sensitive to changes in the lipid domain. Increasing the Choi concentration further caused a redistribution of HP into the lipid domains and phase transitions were observed. The HP redistribution is attributed to a shift in Choi distribution in DPPC. As expected and observed, further increase in Choi concentration (55%) results in total inhibition of phase transitions in DPPC due to increased rigidity. These studies show that photosensitizers such as HP and PP are important probes in liposomes and mixed liposome systems. 

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. 

A handy method is fluorescence polarization measurement. As shown in Fig. 3, however, fluorescence is depolarized by both fluorophore motion and energy migration. Consequently, the method provide qualitative but definitive answer only when... 

Figure 4 General theory of fluorescence polarization. When the smaller partner (that is attached to a fluor) is bound to the larger partner, the emitted fluorescence (red arrows) maintains most of its directionality (polarization) throughout emission (left side). In contrast, when an inhibitor prevents binding, the smaller partner rotates out of alignment during the excited state lifetime, resulting in depolarized emission (light side).

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