Horizontally polarized excitation

For horizontally polarized excitation light, and Ihh are the intensities of the vertically and horizontally polarized fluorescence emissions, respectively. It is strongly recommended to use the background-corrected intensity values for the calculations. The background correction is determined by subtracting the intensity value obtained for the buffer with a certain polarizer setting from the intensity value for the sample obtained with identical settings. 

The G-factor is easily measured using horizontally polarized excitation. With horizontally polarized excitation, the excited-state distribution is rotated to lie along the observation axis. When this is done, both the horizontally and vertically polarized components are equal and proportional to 7x (Figure 10.9). This is because both polarizer orientations are perpendicular to the polarization of the excitation. Hence, any measured difference in Ihv d Ihh must be due to the properties of the detection system. Specifically,... 

The basis for the correction ratio K is that, for horizontally polarized excitation of isotropic samples, the resulting vertically and horizontally polarized fluorescences detected should be of equal intensity any difference may be attributed to the polarization dependence of the emission monochromator and detector. 

Detection limits for various elements by TXRF on Si wafers are shown in Fig. 4.13. Synchrotron radiation (SR) enables bright and horizontally polarized X-ray excitation of narrow collimation that reduces the Compton scatter of silicon. Recent developments in the field of SR-TXRF and extreme ultra violet (EUV) lithography nurture our hope for improved sensitivity down to the range of less than 10 atoms cm ... 

Now, for a given angle 9, excitation can be considered as the superimposition of two beams, one vertically polarized with a weight of cos2 9, and the other horizontally polarized with a weight of sin2 9. Therefore, the intensity components are... 

Fig. 9 Electropherograms showing 3 nM fluorescently labeled 11-mer in the absence (A) and in the presence (B) of 0.7 /j,M SSB protein in the running buffer. The conditions used were as follows separation capillary, 35 cm, 20-/xm i.d. running buffer, 25 mM disodium tetraborate (pH 9.1) separation voltage, 25 kV excitation wavelength, 488 nm emission wavelength, 515 nm and temperature, 25 (1°C. Approximately 1 nL of sample solution was injected electrokinetically. The asterisk indicates the migration time of the solvent, The traces Iv and Ih, corresponding to vertically and horizontally polarized fluorescence intensities, respectively, are shown separated for clarity. (From Ref. 48.)...

Figure B3.6.12 Depolarization of fluorescence indicates rotation of the chromophore. Monochromatic radiation from the source (S) has all but the vertically polarized electric vector removed by the polarizer (P). This is absorbed only by those molecules (see Fig. B3.6.5) in which the transition dipole of the chromophore is aligned vertically. In the case where these molecules do not rotate appreciably before they fluoresce ( no rotation"), the same molecules will fluoresce (indicated by shading) and their emitted radiation will be polarized parallel to the incident radiation. The intensity of radiation falling on the detector (D) will be zero when the analyzer (A) is oriented perpendicular to the polarizer. In the case where the molecules rotate significantly before fluorescence takes place, some of the excited chromophores will emit radiation with a horizontal polarization ( rotation ) and some with a vertical polarization. Finite intensities will be measured with both parallel and perpendicular orientations of the analyzer. The fluorescence from the remainder of the excited molecules will not be detected. The heavy arrows on the left of the diagram illustrate the case where there is rotation.

Table 6 Photocounts of Representative 20 Fluorescent Spots Under the Irradiation of Vertically (V) and Horizontally (H) Polarized Excitation...

In Eq. (14.1), I is fluorescent intensity the subscript letters, V for vertical and H for horizontal, represent the polarization direction of the two polarizers on the excitation and emission light path, respectively and the ratio, ZHV/IhH) calibrates for the difference in the emission channel s sensitivity towards vertical and horizontal polarized components. Anisotropy, r, can be measured by either L-format or T-format. In the L-format, all four fluorescence intensities, Zw, h11, f iv. and ZHh> are measured using a single channel of a photodetector so that each intensity needs to be measured separately. If the fluorimeter has two emission channels then anisotropy can also be measured in a T-format, which allows fluorescence intensities pairs, Ivv//Vi i or If iv // ii i, to be measured simultaneously via the two emission channels. Thus, measurements in the T-format are faster than in the L-format. 

Fig. 26.8. Fluorescence-excitation spectrum from an individual RC-LHl complex from Rps. palustris as a function of the polarization of the excitation light, (a) Top Stack of 312 individual spectra recorded consecutively. Between two successive spectra the polarization of the incident radiation has been rotated by 6.4°. The horizontal axis corresponds to the photon energy, the vertical axis to the scan number or equivalently to the polarization angle and the intensity is given by the gray scale. The excitation intensity was 10 W/cm. Bottom Spectrum that corresponds to the average of the 312 consecutively recorded spectra, (b) Top Fluorescence intensity of the three bands marked by the arrows in the lower part as a function of the polarization of the incident radiation (dots) together with cos -type functions fitted to the data black). Bottom Two fluorescence-excitation spectra from the stack that correspond to mutually orthogonal polarization of the excitation light. The spectra where chosen such that the horizontal polarization yielded maximnm intensity for the narrow feature at the low-energy side. Adapted from [62]...

The originally doubled, Nd-YAG (2 x Nd-YAG) laser beam at 532.0 nm is directed through a KG-3 filter to remove any 1.06-um energy. Part of this laser beam is used to excite the dye in the dye-laser amplifier cell. Usually about 30% of the originally doubled ND-YAG beam is used for dye pumping and the residual 70% is directed to the CARS test section. The dye amplifier cell (DC) is at Brewster s angle to maintain the horizontal polarization of the dye-laser beam. 

Pigure 5.31 shows the experimental Doppler profiles for CO J) products from the photodissociation of OCS with 222 nm laser light. The interesting feature is that horizontal (0 =0) and vertical (0 = 90°) laser polarization does not give the profiles expected for uncorrelated distributions. Por instance, the profile for the Q(58) transition appears to have a P value of about 0.8 when measured with horizontal polarization, while it has a P value of slightly less than 0 when measured with vertical polarization. Purthermore, the Doppler profiles are completely different in the P(58) transition even though the same initial state (J = 58) is being excited. 

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.

In the n) nmasureinents, one chedas for a sensitivity to polarization by excitation with horizontally polarized light The measured values of the differential polarized phase angle (AJ should be zero. Also, the measured value of the modulation ratio (A,) should be 1.0. If needed, FD anisolropy decays can be measured m a T-format to avoid rotating the emission polarizer."... 

The dependence of the radiation pattern from dipoles uniformly distributed within a spherical particle of relative refractive index m = n /n2 = 2.0 upon the particle size is illustrated in Fig.4.9 where the angular distribution in the scattered intensity is plotted in arbitrary units for different values of the size parameter. The exciting radiation incident upon the particle is assumed to be horizontally polarized. There is a sharp increase in the differential scattering... 

Both excitation and emission polarizers could be adjusted to transmit either vertically (0°) or horizontally (90°) polarized light. The two monochromators had different transmission eflSciencies for the vertically and horizontally polarized light that modified the actual measured intensities. This difference in sensitivity is commonly referred to as the G factor and must be determined in order to obtain polarization measurements. We employed the method of Lakowicz to obtain the G factor and to calculate polarization values. 

Fig. 5 77K Polarized excitation spectrum of low-light membranes Detection at 915 nm. Top curve vertical detection, lower curve horizontal detection.

The exciting light is horizontally polarized and is incident on the sample at an angle 0. The intensities of the horizontally (ly) and vertically (ly) polarized emission are measured at an angle tp to the normal of the sample plane. 

FIGURE 1. Experimental geometry of an angle - resolved fluorescence depolarization experiment. Excitation is horizontally polarized and incident on the sample at an angle B, Emission is measured at an angle (p to the membrane director. 

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