Emission channeling

Some particles sputtered from the surface are neutral whereas others are charged. Molecular particles can be emitted either as intact molecules or fragmented. The probability of the desorption of A into the emission channel X is given by the transformation probability P (A -> X ) ... 

Relating back to Eq. (7.8) from the main text, the sensitized emission gray scale image / is composed of the emission from ENs, which depends on the acceptor quantum yield QA, scaled by factors for sensitized emission channel gain (gs), fraction of acceptor fluorescence in the sensitized emission channel F, and donor excitation efficiency tdxD ... 

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. 

Anisotropy measurements yield information on molecular motions taking place during the fluorescence lifetime. Thus, measuring the time-dependent decay of fluorescence anisotropy provides information regarding rotational and diffusive motions of macromolecules (Wahl and Weber, 1967). Time-resolved anisotropy is determined by placing polarizers in the excitation and emission channels, and measuring the fluorescence decay of the parallel and perpendicular components of the emission. 

Another important application of selective extra absorption is the use of chemical shifts in the various X-ray emission channels. This opportunity yields the possibility of measuring the valence-selective X-ray absorption spectra, both XANES and EXAFS. This technique has been applied successfully to Fe(III) as a test case (Glatzel et al., 2002). At least in principle, many applications of this methodology are possible. A metal will have a different X-ray emission spectrum than an oxide. This difference can be used to measure the X-ray absorption spectra of the metal and the oxide in a mixed metal oxide. Such mixtures are encountered frequently in heterogeneous catalysis. 

Fluorescence anisotropy measurements can also be used to obtain the rates of the excited state tautomerization. Two variants can be applied. The first is based on the analysis of time-resolved anisotropy curves. These are constructed from measurements of the fluorescence decay recorded with different positions of the polarizers in the excitation and emission channels. The anisotropy decay reflects the movement of the transition moment and thus, the hydrogen exchange. For molecules with a long-lived Sj state, the anisotropy decay can also be caused by rotational diffusion. In order to avoid depolarization effects due to molecular rotation, the experiments should be carried out in rigid media, such as polymers or glasses. When the Sj lifetime is short compared to that of rotational diffusion, tautomerization rates can be determined even in solution. This is the case for lb, for which time-resolved anisotropy measurements have been performed at 293 K, using a... 

Plate cells at a density of 1 X 10 —2 X 10 cells per well in 100 pi complete growth medium with serum in black walled, clear bottom 96-well plates for tissue culture. Since complementation of click beetle luciferase fragments produces less bioluminescence than intact CBGN or red, we typically use 96-weIl rather than 384-weU plates for cell-based assays. This approach allows us to use shorter acquisition times in each emission channel and improves resolution for kinetics of signahng. For 384-weU plate assays, we use 3 x 10 —5 X 10 cells perweU. We typically culture cells for two days in 96-weU plates before assays. 

Day 2 Transfect the cells. Using the polyethyleniinine method, we use a total of 2 ag of DNA per well. Optimal donor and acceptor plasmids quantities should be determined independently (Sections 2.3.1 and 2.3.2). They must always be complemented with empty vector as a DNA carrier to reach 2 ag/well, to equalize transfection efficiency. Always run also a donor-only control transfection, where the energy donor construct is transfected alone, to measure spectral spillover of the donor emission into the acceptor emission channel. Incubate overnight. 

Figure 1 Determination of optimal ludferase quantity and background BRET. Luciferase titrations are plotted as a function of the amount of transfected energy donor DNA, in the absence of energy acceptor (fluorescent protein). The resulting BRET ratio (black circles) is plotted on the left Y axis, and the luminescence counts (white circles) are plotted on the right Y axis. The detected BRET ratio decreases to reach a background value that is stable over increasing luminescence, and that must be deduced from experimental BRET ratios to obtain BRETnet- This instrument-dependent stable background BRET is due to bleeding of the donor emission into the acceptor emission channel (note the scale difference between the BRET and BRET systems). The higher BRET ratios at too low luminescence counts are due to noise detection in the acceptor emission channel.

The third step was to apply the multicomponent analysis to the smoothed data. The basic principles of this analysis are as follows at each point in time, the measured fluorescence signal in each of the four emission channels is assumed to be a superposition of the fluorescence emission from the different amounts of the four dyes present at that moment in the detection region of the gel tube. These measured values at each time point may be considered to be the components of a four-vector in "detector space". We wish to use these values to deduce the components of a second four-vector a in "dye space", whose components are the different amounts of each dye present in the detector region at that moment. This transformation is represented in equation 1. 

Table III. Some Average Product Translational Energies, Reactive Cross Sections and the Ratios between CH, Emission and H Emission Channels (34, 35). 

---