Emission phase-angle shift

Theory. If two or more fluorophores with different emission lifetimes contribute to the same broad, unresolved emission spectrum, their separate emission spectra often can be resolved by the technique of phase-resolved fluorometry. In this method the excitation light is modulated sinusoidally, usually in the radio-frequency range, and the emission is analyzed with a phase sensitive detector. The emission appears as a sinusoidally modulated signal, shifted in phase from the excitation modulation and partially demodulated by an amount dependent on the lifetime of the fluorophore excited state (5, Chapter 4). The detector phase can be adjusted to be exactly out-of-phase with the emission from any one fluorophore, so that the contribution to the total spectrum from that fluorophore is suppressed. For a sample with two fluorophores, suppressing the emission from one fluorophore leaves a spectrum caused only by the other, which then can be directly recorded. With more than two flurophores the problem is more complicated but a number of techniques for deconvoluting the complex emission curve have been developed making use of several modulation frequencies and measurement phase angles (79). 

For single exponential fluorescence decay, as is expected for a sample containing just one fluorophore, either the phase shift or the demodulation can be used to calculate the fluorescence lifetime t. When the excitation light is modulated at an angular frequency (o = 2itv, the phase angle f, by which the emission modulation is shifted from the excitation modulation, is related to the fluorescence lifetime by ... 

In phase-fluorimetric oxygen sensors, active elements are excited with periodically modulated light, and changes in fluorescence phase characteristics are measured. The delay or emission (phase shift, ( ), measured in degrees angle) relates to the lifetime of the dye (x) and oxygen concentration as follows ... 

In phase-modulation fluorometry, the pulsed light source typical of time-domain measurements is replaced with an intensity-modulated source (Figure 10.5). Because of the time lag between absorption and emission, the emission is delayed in time relative to the modulated excitation. At each modulation frequency (to = 2nf) this delay is described as the phase shift (0, ), which increases from 0 to 90° with increasing modulation frequency. The finite time response of the sample also results in demodulation to the emission by a factor m which decreases from 1.0 to 0.0 with increasing modulation frequency. The phase angle (Ow) and the modulation (m, ) are separate... 

Figure 10. Phase-resolved fluorescence emission spectra for the same sample shown in Figure 9 with a narrow detector phase angle region scanned (0 to 14 ). Note that the emission maxima shifts with detector phase angle and the sign of the phase-resolved intensity changes (curve 9.0°). Both these observations are Indicative of the presence of two components. Laser excitation at 457.9 nmj modulation frequency is 100 MHz.

Phase-resolved, phase-modulation, or phase-sensitive lifetime measurements are based on the use of a continuous, sinusoidally modulated excitation source and phase-sensitive detection (Figure 7). The experimentally measured parameters are the modulation (m) and the frequency-dependent phase shift (4 ). The modulation of the excitation is given by bla, where a is the average intensity and b is the modulated amplitude of the incident light. For emission, the modulation is similarly defined, except using the intensities of the emission, BM, relative to the modulation of the excitation, m = B/A)/ b/a). The phase delay or phase angle ( P) is usually measured from the zero-crossing time of the modulated components. For an exponential decay, the fluorescence lifetime Tf can be calculated from the phase shift or... 

The dipole matrix element contains the radial parts Rp and Rf as well as phase shifts dp and df (see Eq. 4.5). In order to obtain information about these properties the photoelectron intensities were determined at a fixed detection angle d = 45° as a function of the rotation of the E-field vector. The spectra in Fig. 4.7 are shown for particular values of a which the maximum and minimum intensities are reached at for the peak 2 at 4.7 eV (4.0 eV in normal emission) with a = 170° and 80° as well as for the feature 1 at 1 eV and structure 3 at 6 eV with a = 140° and 50°, respectively. The intensity values are summarized in Fig. 4.8 (filled diamond Peak 1, open circle Peak 2, filled square Peak 3). The curves for peak 1 and 3 exhibit the same shape which may be caused by emission from orbitals with the... 

Figure 1. (a) Photoemission spectra N(E) for clean Ni(l 11), O = 5.4 eV (full curve), and after exposure to 2.4 L of benzene at room temperature (broken curve). TTie energy of the unpolarized photons was 21.2 eV, and electrons were collected by the energy analyzer over a large solid angle, (b) Adsorbate-induced difference in emission AA ( ). i.e., the difference between the broken and the full curves in (a). AO = -1.4 eV. (c) AA ( ) for a condensed benzene layer formed at 7" = 150 K at a benzene pressure of 4.6 x lO" Pa. AO = -1.6 eV. (d) Gas-phase photoelectron spectra of benzene. The energy scale in (d) has been shifted so that the assigned levels line up with those in (c). Note that the 7t-level is shifted between (b) and (c). (From Ref. 2.)... 

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