Pulse-amplitude-modulated-fluorescence

D.-P. Hader, H. Herrmann, R. Santas (1996). Effects of solar radiation and solar radiation deprived of UV-B and total UV on photosynthetic oxygen production and pulse amplitude modulated fluorescence in the brown alga Padina pavonia. FEMS Microbiol. Ecol., 19, 53-61. 

In recent years, pulse amplitude modulated (PAM) chlorophyll fluorescence associated with the photosystem II (PS II) has become a useful tool for evaluation of photosynthesis [86-90]. In fact, chlorophyll fluorescence can be an... 

Chlorophyll fluorescence was recorded by a pulse amplitude modulated (PAM) fluorometer (Walz). 

Figure 2 represents a schematic drawing of the combined Chi fluorescence and PAS instrumental setup. The fluorescence of the plant leaf was determined with a Pulse Amplitude Modulation fluorometer (PAM chlorophyll fluorometer, Walz, Effel-trich, Germany) as described in (6). Excitation and emission light passed through the fiberoptics, consisting of four separate bundles, which merge... 

Chlorophyll fluorescence was measured using a pulse amplitude modulation fluorometer (PAM, Heinz Walz, Effeltrich, FRG) as described by [7]. The methods and definitions from [7] were employed to calculate the quenching components qQ, qNP and qTot, which are respectively photochemical quenching, non-photochemical quenching and total quenching of variable fluorescence, all corrected for Fo-quenching (qFo). At each light intensity level, the leaf disc was allowed to achieve steady state of 02-production and fluorescence and then Fs, Fv and Fo were measured. 

A infrared gas analyzer and an open steady state system. Chi fluorescence was measured at 20 C using a pulse amplitude modulated fluorometer (4). Fluorescence measurements were made after leaves used in the gas exchange studies were dark adapted for 90 min. Changes in the ratio of the maximum variable fluorescence (Fvm) to initial fluorescence (Fo) were evaluated in terms of increases in Fo/chl resulting from uncoupling of PSII antenna (5) and decreases in Fvm/chl due to photoinhibition (6). 

Chlorophyll fluorescence was measured by a pulse amplitude modulation fluorimeter Hansatech. 

Chi a fluorescence is a property exhibited by all photosynthetic organisms due to the essential role of chlorophyll in the structure and function of the photosynthetic apparatus. Typically less than 3% of the absorbed light is ever re-emitted as chlorophyll fluorescence and, at room temperature, the latter primarily emanates from PSII (Krause and Jahns, 2003). Quantification of chlorophyll a fluorescence induction has proven to be an extremely useful tool to assess the structure and ftmction of PSII and the overall process of photosynthesis (Krause and Weis, 1991 Krause and Jahns, 2003). Since reduced Qb is in equilibrium with the reduced PQ pool, the relative reduction state of the PQ pool, also called excitation pressure, can be estimated in vivo as the relative reduction state of Qa, that is [Qa ] / ([Qa] + [Qa ]), which can be conveniently measured in vivo as 1-qP using pulse amplitude modulated... 

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. 

If now the period T of the pulses changes and becomes smaller or greater than the atomic period T] 2> it produces dephasing between the various atomic responses S (t) and blurring of their summation the total fluorescence intensity, in stationary regime, is modulated with a smaller amplitude. The amplitude of the fluorescence modulation passes through a maximum when the excitation frequency a> becomes equal to the atomic frequency 12 practice, it is not necessary to use a pulse excitation it is necessary to use a broad band excitation (Aa)exc 12 and to interrupt periodically the excitation light 13. ... 

Instead of a gated switch in the fluorescence detector for pulsed excitation, one may also use excitation by a cw laser that is phase-modulated with a modulation amplitude of tt (Fig. 9.81). The fluorescence generated under sub-Doppler excitation in a collimated atomic beam is observed during a short time interval A, which is shifted by the variable delay T against the time to of the phase jump. If the fluorescence intensity T) is monitored as a function of the laser frequency cu,... 

This result gives the modulation in a Doppler free form. The requirement is that the line be excited uniformly. The averaging over the velocity groups has introduced an additional Gaussian degradation term which can be rewritten in the form exp- (l/2) [ ( 2 -Q)/Q] (x/ ] is of no consequence in the regime in which we work since 2Xjj 2/(Q-Q ) is of order 400 nsec. For a pulse separation of x=400 nsec the echo which appears 2x =800 nsec or 50 fluorescence lifetimes later would suffer a diminished modulation amplitude of only e . It seems then that this technique can effectively utilize the abiltiy of the photon echo technique to observe excited atoms which live many times their natural lifetime. 

We have already discussed quantum-beat spectroscopy (QBS) in connection with beam-foil excitation (Fig.6.6). There the case of abrupt excitation upon passage through a foil was discussed. Here we will consider the much more well-defined case of a pulsed optical excitation. If two close-lying levels are populated simultaneously by a short laser pulse, the time-resolved fluorescence intensity will decay exponentially with a superimposed modulation, as illustrated in Fig. 6.6. The modulation, or the quantum beat phenomenon, is due to interference between the transition amplitudes from these coherently excited states. Consider the simultaneous excitation, by a laser pulse, of two eigenstates, 1 and 2, from a common initial state i. In order to achieve coherent excitation of both states by a pulse of duration At, the Fourier-limited spectral bandwidth Au 1/At must be larger than the frequency separation ( - 2)/ = the pulsed excitation occurs at... 

To derive the expressions for the fluorescence phase shift (p) and modulation amplitude m) in Fig. 1.16, suppose the oscillatory part of the excitation light intensity is I t) = sin(mO. and that the fluorescence (F t)) generated by an instantaneous pulse of exciting light decays exponentially with time constant n. The oscillatory part of the fluorescence signal observed at time t (S(0) is obtained by integrating the fluorescence from the modulated excitation at all earlier times ( ) ... 

If two or more closely spaced molecular levels are simultaneously excited by a short laser pulse, the time-resolved total fluorescence intensity emitted from these coherently prepared levels shows a modulated exponential decay. The modulation pattern, known as quantwn beats is due to interference between the fluorescence amplitudes emitted from these coherently excited levels. Although a more thorough discussion of quantum beats demands the theoretical framework of quantum electrodynamics [11.33], it is possible to understand the basic principle by using more simple argumentation. 

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