Polarization-analyzed fluorescence

Figure 49. Polarization-analyzed fluorescence decays for S, + 1249 cm-1 excitation of jet-cooled t-stilbene. The fluorescence band detected whs at a shift of —205 cm 1 from the excitation energy and was detected with 32 cm 1 resolution.

Clearly, much activity may be expected in the specific area of rotational coherence and the more general one of time-resolved and polarization-analyzed fluorescence. One area of interest, not touched upon here, concerns the influence of rotational coherence in electronic relaxation processes. In this we regard it is pertinent to note the polarization-dependent decays reported first by Matsumoto et al.72 in their studies on the nanosecond time scale of singlet-triplet coupling in pyrazine. 

Fig. 4.9. Schematic of time-resolved fluorescence anisotropy sample is excited with linearly polarized light and time-resolved fluorescence images are acquired with polarization analyzed parallel and perpendicular to excitation polarization. Assuming a spherical fluorophore, the temporal decay of the fluorescence anisotropy, r(t), can be fitted to an exponential decay model from which the rotational correlation time, 6, can be calculated.

Figure 3-16 Schematic diagram of a fluorescence polarization analyzer. P is the polarizer to provide polarized excitation light. PA is the polarizer analyzer, which is rotated to provide the measurement of parallel and perpendicular polarized fluorescence-emission intensity. ExM is the excitation monochromator, EmM is the emission monochromator, D is the detector, and C is the reaction cell or cuvet.

As indicated, P is the difference between the two observed intensities divided by their sum. Fluorescence polarization is measured by placing a mechanically or electrically driven polarizer between the sample cuvet and the detector. A diagram of a fluorescence polarization measurement system is shown in Figure 3-16. In the normal instrumentation mode, the sample is excited with polarized hght to obtain maximum sensitivity. The polarization analyzer is positioned first to measure the intensity of the emitted fluorescence hght in the vertical plane ly), and then the polarization analyzer is rotated 90° to measure the emitted fluorescence hght intensity in the horizontal plane (Ij,). P is then calculated manually or automatically by use of equation (13). 

Now consider the temporal behavior of polarization-analyzed (polarization vector if) fluorescence from this superposition state to all possible rotational eigenstates of the ground vibronic level ISqPj-). This fluorescence decay is... 

The polarization of fluorescence data below the transition temperature were analyzed according to Equation 1, and the rotational relaxation time, phb, and the equivalent volume of the rotating segment, Ve5, were calculated at 5°C. from the equations ... 

The laser is then fed into the multipass system with the cesium cell in the center. The fluorescence at 1.36/xm is measured in an orthogonal direction. This is passed through a circular polarization analyzer rotating at (Of and then an interference filter to reject oven light and into a Ge photodiode. 

The time-resolved fluorescence anisotropy function, rif), is calculated using Equation 1 in which / (0 and IJJ) are the individual decays collected with the polarization analyzer set parallel and perpendicular to the vertically polarized excitation light. The G factor is included to account for any polarization bias of the detection system. The influence of this term was minimized by arranging the polarization analyzer to be the first element in the detection system and using a polarization pseudo-scrambler (Oriel 28115) immediately prior to the emission monochromator slit. 

A newer and perhaps more useful application of ellipsometry to Langmuir films is their lateral characterization via ellipsometric microscopy [146], A simple modification of the nuU ellipsometer allows one to image features down to 10-/im resolution. Working with a fixed polarizer and analyzer, some domains are at extinction while others are not and appear bright. This approach requires no fluorescent label and can be applied to systems on reflective supports. 

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

Like Raman scattering, fluorescence spectroscopy involves a two-photon process so that it can be used to determine the second and the fourth rank order parameters. In this technique, a chromophore, either covalently linked to the polymer chain or a probe incorporated at small concentrations, absorbs incident light and emits fluorescence. If the incident electric field is linearly polarized in the e direction and the fluorescent light is collected through an analyzer in the es direction, the fluorescence intensity is given by... 

Recently, a formalism has been developed to determine the second and the fourth order parameters of films using polarized total internal reflection fluorescence (TIRF) [71]. Similarly to IR-ATR spectroscopy (Section 4), the experiment makes use of p- and s-polarized excitation, but the fluorescence emission (analyzed either in p- or s-direction) is detected normal to the substrate. Two approaches are developed based on the measurements of two intensity ratios. In the first one, the S angle has to be known experimentally or theoretically, and the order parameters (P2) and (P4) can be determined. In the second one, the order parameter (R ) is obtained by another technique, for instance IR-ATR spectroscopy, which allows deducing the order parameter (P4) and (cos2<5). 

Five anticonvulsants including valproic acid were determined by the Abbott TD x fluorescence polarization immunoassay automatic analyzer. Recoveries were 94.8-106% and the coefficients of variations were 1.0-9.7% [23], Fluorescence polarization immunoassay and enzyme immunoassay were compared for the determination of free valproic acid in serum [24], Good correlation (R = 0.9992) was obtained between the two assays. Higgins [25] reported on the determination of valproic acid in serum by enzyme immunoassay with use of EMIT reagents and the Abbot ABA-200 analyzer. Responses were rectilinear up to 150 mg/L. 

Fig. 1. Typical locations for CAM components, showing the photometer, 1 filter wheel, 2 monochromator, 3 shutter and aperture unit, 4 beam splitter, 5 accessories for polarized light such as a rotary analyzer and a compensator, 6 beam splitter for epi-excitation fluorescence, 7 objective lens, 8 stage, 9 substage condenser, 10 condenser aperture, 11 polarizer, 12 field aperture for photometry, 13 shutter, 14 primary illuminator, 15 arc lamp, 16 shutter, 17 monochromator, 18 filter wheel, 19 and ocular, 20.

Fluorescence polarization is also well suited to equilibrium binding studies when the free and bound species involved in the equilibrium have different rotational rates (Scheme 5.1). Most molecular interactions can be analyzed by this method. It should be emphasized that, in contrast to other methods using tracers, fluorescence polarization provides a direct measurement of the ratio of bound and free tracer without prior physical separation of these species. Moreover, measure-... 

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