Light demodulation

The fluorescence lifetime of the sample can be calculated from phase delay and/or from light demodulation. The longer the sample lifetime, the more the emission is delayed and greater the phase shift b ween the two lights. 

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 ... 

Luminescence lifetimes are measured by analyzing the rate of emission decay after pulsed excitation or by analyzing the phase shift and demodulation of emission from chromophores excited by an amplitude-modulated light source. Improvements in this type of instrumentation now allow luminescence lifetimes to be routinely measured accurately to nanosecond resolution, and there are increasing reports of picosecond resolution. In addition, several individual lifetimes can be resolved from a mixture of chromophores, allowing identification of different components that might have almost identical absorption and emission features. 

To analyze frequency domain FLIM data, first the phase shift and demodulation of the fluorescence light with respect to the excitation light are estimated. In the case of single frequency data, this reduces the FLIM data to only three parameters phase shift, demodulation, and total intensity. This step can be done in various ways as described in the following sections. From these parameters, the lifetimes can be estimated either by Eqs. (2.6 and 2.7), or by more elaborate approaches as described below. 

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. 

Prior to describing the possible applications of laser-diode fluorometry, it is important to understand the two methods now used to measure fluorescence lifetimes these being the time-domain (Tl)/4 5 24 and frequency-domain (FD) or phase-modulation methods.(25) In TD fluorometry, the sample is excited by a pulse of light followed by measurement of the time-dependent intensity. In FD fluorometry, the sample is excited with amplitude-modulated light. The lifetime can be found from the phase angle delay and demodulation of the emission relative to the modulated incident light. We do not wish to fuel the debate of TD versus FD methods, but it is clear that phase and modulation measurements can be performed with simple and low cost instrumentation, and can provide excellent accuracy with short data acquisition times. 

The luminescence L(t) contains only a subset of modulation frequencies of the excitation light due to the demodulation at higher frequencies caused by the molecule decay rates (see Eq. (9.51)). This subset is given by... 

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

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... 

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. 

As discussed earlier, the PEM is, in many ways, an ideal modulation device for polarization-selective measurements. Thus, the optimum simultaneous multiwavelength CD instrument should incorporate both the PEM and CCD, and at the same time allow the PEM to operate at its resonant frequency. One way to overcome the basic incompatibility of the PEM and CCD is to use an optical demodulation scheme in which the two oppositely polarized components would be directed by a polarizing beamsplitter, to either different areas of the same CCD, or to different CCDs [14]. Since two distinct detector areas would be accessed by the oppositely polarized light, pixel-to-pixel sensitivity variations may be a significant source of noise. 

We detected the saturated fluorescence emitted by a beam of 23S metastable atoms as they cross at right angle the slave laser light. A 1015 atoms/s.sterad flux of metastable helium atoms was produced by electronic collisions in a DC discharge of a helium atomic beam, similar to that described in [15]. To improve the precision of the linecenter determination, we increased the signal-to-noise ratio S/N by means of standard frequency modulation the third harmonic demodulated lineshape is shown in Fig. 4. The function expected for a Lorentzian spectrum was fit and linecenters were calculated with an uncertainty ranging between 10 kHz and 20 kHz, that is consistent with the observed S/N, mainly limited by the stability of the reference frequency and of the metastable helium beam. The reproducibility was two or three times worse than the uncertainty,... 

The basic optical setup was shown in Fig. 12 [90]. The spectra were recorded on a commercially available spectrometer equipped with an external PM setup. The photoelastic modulator modulated the polarization of the IR light at a fixed frequency. Demodulation was performed with a lock-in amplifier and a low-pass filter. After the IR beam passes through the polarizer and modulator, it is focused on the sample, then focused on an mercury-cadmium-telluride (MCT) detector cooled by liquid nitrogen. 

The differential absorption information is recovered using phase sensitive detection techniques. The demodulated absorption line shape is shown in Fig. 4. The amount of light absorbed is directly proportional to oxygen concentration. The gas density, n, is related to the peak-to-peak signal amplitude. A/, by Beer s law, which for a weakly absorbing molecule, is given by ... 

Modems (MODulator/DEModulators) are the devices that computers use to talk to one another over phone lines. They can be considered a type of output device because they move data out of the computer to another device. Modems work by converting digital signals (binary Is and Os) into analog signals (tones over a phone line), and vice versa. Modems are added to a computer either as an external device or as an expansion card installed inside the computer. Internal modems are usually less expensive than external modems, but external modems are easier to troubleshoot than internal modems because you can see the lights that indicate what is happening. Lor more information on modems, see Chapter 6, Peripheral Devices. ... 

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. 

A glycogen solution placed in the emission compartment will scatter light and be used as reference (tf = 0) to determine phase delay and fluorescence demodulation For each measurement, the intensity of the reference is adjusted in order to have it equivalent to the intensity of the fluorescence signal of the sample. Phases and modulations of the fluorescence and scattered light are obtained relative to the reterence photomultiplier or instrumental (internal) reference signal. In fact, two Identical detection electronic systems are used to analyze the outputs of the reference and sample photomultipliers. Each channel consists of an alternative and continuous cunents (AC and AD, respectively). 

In the frequeney domain, the sample is exeited with modulated light (D). The amplitude and the phase are measured at a single frequeney or at a small number of frequeneies. Different modulation frequeneies ean be obtained by ehanging the exeitation frequeney or by using different harmonies of a pulsed exeitation waveform. The effieieney of the modulation teehnique depends on a number of techni-eal details, espeeially the depth of modulation of the exeitation light and the way the deteetor signal is demodulated. Only for exeitation with short pulses of high repetition rate and ideal demodulation a near-ideal effieieney is obtained. 

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 ... 

Examination of Hgure 4.2 reveals another effect of the lifetime, tiiis bang a decrease in the peak-to-peak bright of the emission relative to that of the modulated excitation. The modulation decreases because some of the fluoto-phores excited at the peak of the excitation continue to emit when the excitation it at a minimum. The extent to which diis occurs depends on the decay time and light modulation frequency. This effiect is called demodulation and can also be used tocalculate the decay time. At present, pulse and phase-modulation measurements ate both in widespread use. 

Figure 10.13 shows experimental setup for the optical characteristic measurement of PMNT ceramics [133]. The size of PMN-PT ceramic sample was 5 mm X 2 mm x 1 mm for length x width x thickness. Ti/Pt/Au layers were sputtered on both surfaces of the ceramics as electrodes. Two collimators were used to collimate the incident beam and receive the transmission beam. The output beam was detected by using an optical spectrometer and phase demodulation. Because the PMN-PT electro-optic ceramics have a large refractive index, i.e., n = 2.465, the ceramic samples could be considered as a Fabry-Perot (FP) resonator, which can be used to measure the electric hysteresis and thermo-optic coefficient. The applied voltage generated a transverse electro-optic effect for the transmission light beam. 

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