Sinusoidally modulated light

Phase-modulation fluorometry The sample is excited by a sinusoidally modulated light at high frequency. The fluorescence response, which is the convolution product (Eq. 6.9) of the pulse response by the sinusoidal excitation function, is sinusoidally... 

In the frequency domain, any periodic excitation, r.(t), can be described by a sum of sinusoidally modulated light waveforms at harmonics of the fundamental frequency of the excitation... 

Each sinusoidally modulated light intensity of the response is phase delayed and demodulated with respect to the excitation such that... 

Phase-modulation immunoassay measurements are made with sinusoidally modulated light. Since the emission is a forced response to the excitation, the emitted light has the same periodicity as the excitation. Due to the time lag between absorption and emission, the emission is delayed in comparison with the excitation. The time delay between the zero crossing of one period of the excitation and of the emission is measured as the phase angle (Figure 14.11). The emission is also demodulated, due to a decrease in the alternating current (AC) component of the AC to direct current (DC) ratio. 

It is possible to resolve the emission from one species in the presence of another by phase-suppression techniques. The emission from a single fluorophore excited by sinusoidally modulated light is described by... 

The measuring technique consists of exciting the sample with sinusoidally modulated light and determining either the depth of modulation or the phase shift of the fluorescent emissions. 

Fig. 8.1. Schematic illustration of the TG experiment [upper) and the principle of diffusion measurement [lower). Lower The white and black circles indicate the reactant and product molecules. The concentrations of the reactant and the product are spatially modulated by the sinusoidally modulated light intensity of the grating light. The fringe length A is also indicated...

In phase-modulation fluorometry, the sample is excited by a sinusoidally modulated light at high frequency. The fluorescence response, which is the convolution product (Eq. (7.6)) of the d-pulse response by the sinusoidal excitation function, is sinusoidally modulated at the same frequency but delayed in phase and partially demodulated with respect to the excitation. The phase shift and the modulation ratio M (equal to m/mo), that is the ratio of the modulation depth m (AC/DC ratio) of the fluorescence and the modulation depth of the excitation mg, characterize the harmonic response of the system. These parameters are measured as a function of the modulation frequency. No deconvolution is necessary because the data are directly analyzed in the frequency domain. 

Lifetimes. The theory of frequency-domain lifetime determinations has been described in detail elsewhere (15-19). Briefly, a high-frequency (MHz - GHz) sinusoidally-modulated light source is used to excite the fluorescent sample. The time-dependent mathematical representation of the excitation waveform (Ex(t)) is given by ... 

With frequency domain FLIM the light source is a continuous wave laser as opposed to a pulsed laser. The continuous wave laser is modulated via an acousto-optical modulator and the sample is excited by a sinusoidally modulated light. The fluorescence response is also sinusoidally modulated at the same frequency but it is delayed in phase and is partially demodulated. For a single exponential decay the lifetime of the donor chromophore can be quickly calculated by either the phase shift (j) (rp) or the modulation ratio M (r ,) using the following equations ... 

In a phase-modulation fluorometer, the sample is illuminated with sinusoidally modulated light which is intensity-modulated with a circular modulation frequency (i> (Hgure... 

Another technique recently applied for immunoassays is phase-resolved fluorometry (frequency-domain fluorometry). This technique is also based on different fluorescence decay times. The decay time can change upon antigen-antibody binding. Instead of pulsed excitation, in this technique the sample is excited with sinusoidally modulated light. With phase-resolved fluorometry, decay times and decay time differences within the range of subnanoseconds can be measured. The phase-resolved technique can be used also for elimination of background noise. This technique, however, has found only a few applications to immunoassays yet. 

Photocurrent transient methods [44, 150-155] and intensity-modulated photocurrent spectroscopy (IMPS) [142, 151, 156-161] have been established as good techniques for the investigation of semiconductor surfaces [162]. By these methods the working electrode is illuminated with either a light pulse or with sinusoidal modulated light, respectively, and the resulting photocurrent is monitored. Both techniques have been used at phthalocyanine electrodes to analyze in detail the kinetics of the photoelectrochemical reactions. 

Phsise-Shift Method. In the phase-shift method atoms or molecules are excited by a sinusoidally modulated light or electron beam, as illustrated in Fig. 9.27. Fluorescence light is recorded with a photomultiplier tube. This light will thus jiilso be modulated, but because of the delay in the excited state a phase-shift is introduced [9.149]. At the same time, the contrast in... 

Fig. 1.16 (A) Fluorescence (so/id curve) from a molecule that is excited with sinusoidally modulated light (dotted curve). If the fluorescence decays exponentially with single time constant T, the phase shift (4>) and the relative modulation of the fluorescence amplitude (w) are related to t and the angular frequency of the modulation m) by < = arctan((OT) and w = (1 + o> ) (Appendix A4). The curves shown here are calculated for t = 8 ns, cd = 1.257 x 10 rad/s (20 MHz) and 100% modulation of the excitation light (< = 0.788 rad, i = 0.705). (B) Phase shift (4>, solid curve) and relative modulation (m, dotted curve) of the fluorescence of a molecule that decays with a single exponential time constant, plotted as a functirai of the product on. The relationships among , m, r and m become more complicated if the fluOTescence decays with multiexponential kinetics (Appendix A4)...

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