Depolarization measurement

Depolarization measurements, coupled with fluorescence lifetimes, are correlated with rates of molecular rotation to obtain estimates of molecular conformation, volume, and shape. 

Any factor that affects the size or shape of a molecule, the hindered movement of a fluorophore within a molecule, or the energy transfer within the molecule will affect the measured depolarization of its fluorescence emission. Therefore, the conformation of humic fractions in solution can be studied as a function of pH, ionic strength, temperature, and other factors by depolarization measurements. The principle of the method is that excitation of fluorescent samples with polarized light stimulates... 

A depolarization measurement consists of exciting a fluorescent sample with linearly polarized light and measuring the polarization of emitted light at right angles to the plane of excitation. The polarization of the emitted light is defined as... 

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 ... 

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.

T. A. M. Doust, Fluorescence lifetime and depolarization measurements using frequency up-con-version, in Picosecond Chemistry end Biology (T. A. M. Doust and M. A. West, eds.), pp. 1-34, Science Reviews Ltd, London (1983) and references therein. 

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... 

Figure 2-1 of Chapter 2 shows an experimental configuration for depolarization measurements in 90° scattering geometry. In this case, the polarizer is not used because the incident laser beam is almost completely polarized in the z direction. If a premonochromator is placed in front of the laser, a polarizer must be inserted to ensure complete polarization. The scrambler (crystal quartz wedge) must always be placed after the analyzer since the monochromator gratings show different efficiencies for L and polarized light. For information on precise measurements of depolarization ratios, see Refs. 21-24. 

The general approaches used in the studies considered below for assignment of the observed vibrational bands to the short-lived molecules are analogous to those described in Sections III and IV. The assignment of the revealed bands to normal, or fundamental, vibrational modes has been based on taking into account selection rules, observations of the bands in characteristic regions, observations of isotopic shifts, results of depolarization measurements in the Raman spectra and results of normal coordinate analysis. (It is noteworthy that Raman depolarization measurements can be conducted for matrix isolated species as well see Reference and references cited therein.) Lately, quantum-chemical calculations of vibrational spectra have become an important tool for both identification of CAs and assignment of their vibrational spectra. 

Since, in HRS, there is no preferred orientation induced by an additional static field, there is the possibility of varying the experimental conditions in order to increase the number of independent observables. The number of theoretically possible independent observations, and hence the number of tensor components that can be obtained by HRS, is at most five. For parametric light scattering, this number is six, due to the possibility of distinguishing between the two optical fundamental fields [20]. The experimental difficulty has precluded the determination of this number of components. What is experimentally realistic in HRS is an additional depolarization measurement, apart from the classical measurement of the intensity of the second-order incoherent scattered light. The two measurements, the total intensity measurement and the depolarization ratio (or two intensity measurements, one with parallel and one with perpendicular polarization for fundamental and second harmonic), represent two independent observables and allow the experimental determination of two tensor components. For molecules of C2 symmetry, these are and P xxy resulting for the total intensity measurement in Eqn. (21),... 

Fig. 44. Experimental geometry for time-resolved flimescence depolarization measurements...

Thus, as mentioned earlier, time-resolved depolarization measurements afford a means of recording the time profile of the rotational autocorrelation function. The steady state technique, with continuous sample excitation, produces merely the time average of the emission anisotropy, F. For a rotating chromc hore with a sin e fluorescence decay time Tf, F is related to r(t) by the following expression... 

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

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