Probes excitation, emission spectra

Knowledge on the plasma species can be obtained by the use of plasma diagnostics techniques, such as optical emission spectroscopy (OES) and mass spectroscopy (MS). Both techniques are able to probe atomic and molecular, neutral or ionized species present in plasmas. OES is based on measuring the light emission spectrum that arises from the relaxation of plasma species in excited energy states. MS, on the other hand, is generally based on the measurement of mass spectra of ground state species. 

An increase in sensitivity and reliability of chip analysis can also be achieved by using fluorescence resonance energy transfer (FRET). For this purpose both the probe and the target are labeled with a fluorophor. When the emission spectrum of the donor, e.g. Cy5, overlaps with the absorption spectrum of the acceptor, e.g. Cy5.5, and the donor and the acceptor are at a certain distance from each other, energy is transferred from the donor to the acceptor on excitation of the donor fluorophor. 

In principle, the problems of intensity-based sensing can be avoided using wavelength-ratiometric probes, i.e., fluorophores that display spectral changes in the absorption or emission spectrum on binding or interaction with the analytes (Figure 1.1). In this case, the analyte concentration can be determined independently of the probe concentration by the ratio of intensities at two excitation or two emission wavelengths. 

If the probe display shifts in absorption and/or emission spectra, the apparent dissociation constant will depend not only on the measured parameter but also on the excitation and emission wavelengths. For probes that display a shift in the absorption spectrum on analyte binding, the value of f/ bdepends on excitation wavelength, which affects the value of Koa (see Eq. (10.24)). For probes which display shift in emission spectrum the value of may depend on observation wavelength. The... 

The first term in Eq. (4.3) is reminiscent of Eq. (3.2) for the spontaneous emission spectrum. It represents a doorway wavepacket created by the pump in the excited state, which is then detected by its overlap with a window. The only difference is that the role of the gate in determining the window in SLE is now played by the probe Wigner function W2. In addition, the pump-probe signal contains a second term that does not show up in fluorescence. This term represents a wavepacket created in the ground state (a hole ) that evolves in time as well and is finally determined by a different window Wg [24]. In the snapshot limit, defined in the preceding section, we have... 

Eu + ion was often used as a probe to detect the crystal environment in which the ion is located. In LaP04, Eu can be excited at 260 run because of a charge transfer band caused by the electron transfer in the Eu +-0 bond. In the emission spectrum of Eu " ", the ratio of the different peaks of the Fj (/ = 1, 2) transitions gives information about the symmetry of the crystal site in which Em is located, which is Ci in the monazite type LaP04, the same as in the bulk materials (Stouwdam et al., 2003). 

In addition to parinaroyl phospholipids, pyrene fatty acid derivatives may be used. Such phospholipids have a concentration-dependent emission spectrum (Roseman and Thompson, 1980). At low concentrations of the derivative within the bilayer, the fluorescence is maximal at a wavelength below 400 nm. At higher concentrations of the derivative, the excited state monomers can associate with a ground state monomer to form a dimer complex, or eximer, in a diffusion-controlled process. The maximum emission wavelength of the eximer shifts to approximately 470 nm. The ratio of the eximer to monomer fluorescent intensity is proportional to the concentration of the probe molecules within the bilayer. 

In leccni years, it has become possible to combine fluorescence spectroscopy with optical microscopy to produce localized images of fluorophores in complex matrices such as single cells. In some cases the intrinsic (native) lluoresccnce of biomoiccules can be used in conjunction with microscopy to monitor dynamics in cells.In the absence of a native fluorophore, fluorescent indicators can be used to probe biological events. A parlicularlv interesting probe is the so-called ion probe that changes its excitation or emission spectrum on binding lo specific ions such as Ca or Na. These indicators can he used to record events that take place in different parts of individual neurons or lo monitor simultaneously the activity of a collection of neurons. 

Figure 1- Emission spectrum for Tm -Yb codoped oxyfluoride glass ceramic associated to the G4 —> p4 electronic transition. The solid line gives the emission of the sample under pump excitation and the dashed line shows the emission under pump and probe excitation. The signal wavelength is indicated with an arrow...

Fluorescence studies 14, 15) using pyrene, pyrene derivatives, and cationic probes in poly(methacrylic acid) have shown that a conformational transition from a closed compact coil to extended form induced by pH is a progressive process over several pH units (pH 4-6). The emission spectrum of 4 X 10 M R6G and 4 X 10 M RB excited at 480 nm in water is not dependent on pH. However, in aqueous solutions of PM A, the spectra are significantly dependent on pH (shown in Figure 6). At pH 4-5, the spectra are similar to the typical emission of RB at pH 2-3 and 6-7, the spectra in PMA display stronger emission at 550 nm and at pH 8, the spectra are identical to those in water. 

Effect on anission maximum (X ax) The effect can be used to estimate polarity of the environment around the fluorophore. In general, the excited state is more polar than the ground state. Thus the excited molecules tend to interact with a polar environment favorably and cause the red shift in the emission spectrum. However, an orientation constraint on the fluorophore may cause a blue shift if the probe molecules do not have time to undergo rearrangement. 

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