Modulated excitation

Pulsed method. Using a pulsed or modulated excitation light source instead of constant illumination allows investigation of the time dependence of emission polarization. In the case of pulsed excitation, the measured quantity is the time decay of fluorescent emission polarized parallel and perpendicular to the excitation plane of polarization. Emitted light polarized parallel to the excitation plane decays faster than the excited state lifetime because the molecule is rotating its emission dipole away from the polarization plane of measurement. Emitted light polarized perpendicular to the excitation plane decays more slowly because the emission dipole moment is rotating towards the plane of measurement. 

At present, two main streams of techniques exist for the measurement of fluorescence lifetimes, time domain based methods, and frequency domain methods. In the frequency domain, the fluorescence lifetime is derived from the phase shift and demodulation of the fluorescent light with respect to the phase and the modulation depth of a modulated excitation source. Measurements in the time domain are generally performed by recording the fluorescence intensity decay after exciting the specimen with a short excitation pulse. 

Sanden, T., Persson, G., Thyberg, P., Blom, H. and Widengren, J. (2007). Monitoring kinetics of highly environment sensitive states of fluorescent molecules by modulated excitation and time-averaged fluorescence intensity recording. Anal. Chem. 79, 3330-41. 

Another way the SR might modulate excitability in the uterus is via Ca2+-activated Cl- (ClCa) channels. The evidence for ClCa in the uterus is limited but Arnaudeau et al (1994b) showed that oxytocin in rat cells appeared able to stimulate these channels, and we also find their activation via voltage-gated Ca2+ entry (K. Jones, A.V. Shmigol S. Wray, unpublished observation). 

Continuous wave techniques do not offer the optimum use of luminescence for sensing applications. CW methods, also known as intensity-based techniques, have many inherent limitations. These limitations will be discussed later in the chapter. Many of the limitations of intensity-based methods can be overcome by using steady-state modulated excitation of the form... 

The use of high-speed modulated excitation (f> kr + knr) combined with coherent detection methods has resulted in the popular techniques of frequency domain fluorometry, also known as phase-modulation fluorometry. These techniques can be used to determine the temporal characteristics of both fluorescence and phosphorescence and will also be addressed later in this chapter. 

The transient response of luminescent substances to modulated excitation can be determined in the frequency domain by measuring the phase delay and the demodulation of the luminescence with respect to the excitation. (14,23 28)... 

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 14.11. Diagram of phase-modulation fluorometry with sinusoidally modulated excitation, with demodulated and delayed, or phase-shifted emission. (From Ref. 31 with permission.)...

There are two ways to collect FLIM data freqnency-domain or time-domain data acqnisition (Alcala et al. 1985 Jameson et al. 1984). Briefly, in freqnency domain FLIM, the fluorescence lifetime is determined by its different phase relative to a freqnency modulated excitation signal nsing a fast Fourier transform algorithm. This method requires a frequency synthesizer phase-locked to the repetition freqnency of the laser to drive an RF power amplifier that modulates the amplification of the detector photomultiplier at the master frequency plus an additional cross-correlation freqnency. In contrast, time-domain FLIM directly measures t using a photon connting PMT and card. 

The aim of this review is to provide an assessment of the state of this field. After a summary of some basic theoretical results, the focus is on experimental aspects, ranging from cell design to specialized techniques such as modulation excitation spectroscopy (MES). We emphasize the opportunities and limitations of ATR-IR spectroscopy in catalysis research. 

In the following, we describe such techniques, which are, besides time-resolved spectroscopy, modulation excitation spectroscopy (MES) (14,48,91) and singlebeam signal reference (SBSR) spectroscopy (14). 

The measurement of the growth and decay of fluorescence requires essentially two items (a) modulated excitation source and (b) a detector. The modulation of an excitation source may be accomplished in various ways. These range from simple mechanical choppers to highly sophisticated electronic pulsers. Detectors may be phototubes or semiconducting devices, or even the human eye. The detector itself, in some instances, may be modulated. Of course, the detector chosen must depend upon the spectral range to be studied and the response time desired. 

Farrell, E.F., Antaramian, A., Rueda, A., et al., 2003, Sorcin inhibits calcium release and modulates excitation-contraction coupling in the heart. J Biol Chem, 278, pp 34660-66. 

The reaction Fe ccp/Fe cytc + Fe ccp/Fe cytc proceeds with AE s 0.4V. The reaction h j been moj ored both by pulse radiolysis, and by simple mixing of Fe ccp + Fe1 cytc, with equivalent results k 0.25 0.07 s (figure 10) It is interesting that a dependence of rate on the primary structure of the protein is observed (at constant AG) for horse cy1j.c/ccp(yeast) k = 0.25 s but for yeast. cytc/(yeast) ccp k = 4 s 1 and for tuna cytc/yeast ccp k s 0.1 s, even though the general three dimensional structures are essentially identical for horse, tuna and yeast cytochromes c. These determinations disprove an earlier suggestion based on modulated excitation spectroscopy, that k - 10 s. Clearly the rate is slow,... 

We have implemented the discrimination in the frequency domain. As is known in multifrequency phase fluorometry,17 the time-delayed fluorescence acquires a phase shift >p and a reduction in amplitude Mp upon increasing the modulation frequency m = 2irf of the sinusoidally modulated excitation. For a simple single exponential decay, this phase shift

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Ferri D, Kumar MS, Wirz R, et al. First steps in combining modulation excitation spectroscopy with synchronous dispersive EXAFS/DRIFTS/mass spectrometry for in situ time resolved study of heterogeneous catalysts. Phys Chem Chem Phys. 2010 12 5634. 

One of the most convenient methods for determining lifetime, as well as one of the best suited to low-cost applications, involves using frequency-modulated excitation (49). Upon excitation by a frequency modulated source, the finite lifetime of the emitter causes a phase-shift and demodulation of the emission relative to the excitation waveform as shown schematically in Fig. 6. 

Figure 6. A schematic representation of a modulated excitation wave form (solid line) and the time delayed modulated emission waveform (dashed line). The parameters Atp and AA represent the change in phase and the change in modulation amplitude of the two wave forms.

Reid, M.B., Kobzik, L., Bredt, D.S., Stamler, J.S. (1998). Nitric oxide modulates excitation-contraction coupling in the diaphragm. Comp. Biochem. Physiol. A Mol. Integ. Physiol. 119(1) 211-18. 

Due to the short lifetime of the fluorescence decay process (10 — 10 s) time-resolved fluorescence studies provide a number of experimental difficulties and require the use of more sophisticated apparatus. Several tedini(pes have been applied to measure fluorescence decay characteristics but all require the use of a pulsed or modulated excitation source (see reviews by Birks, Ware, Knight and Selinger ). 

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