126
modulated excitation has two advantages. First, its intensity may be kept low enough that the
measuring source alone produces no actinic effects (such that the modulated source alone
measures only Fo in a dark adapted sample). Use of a lock-in amplifier in the detection
circuitry provides for sufficient signal to noise even at the low excitation intensity. Second,
the modulated fluorescence signal is essentially independent of any direct effects of all nonmodulated sources illuminating the sample. This means that an instantaneous change in the
intensity of a non-modulated source will have no affect on the instantaneous fluorescence
yield. However, the non-modulated sources will have an indirect, actinic effect by altering
the state of the photochemical reactions in the sample. Thus the modulated fluorescence signal
measures a true, relative fluorescence yield. The modulated fluorescence technique and its
advantages are described in detail by Schreiber (1983, 1986).
An additional advantage of the modulated fluorescence measurements is that it provides for
a non-destructive technique for separating photochemical and non-photochemical fluorescence
quenching (Schreiber et aI., 1986). In this procedure, brief (0.1-2 s) illumination with high
intensity light is used to induce a transient, complete reduction of Ch, bringing qp to O. Any
fluorescence quenching remaining when qp = 0 is by definition em (Figure 8). This technique
of "light doubling" was introduced by Bradbury and Baker (1984). The combination of
modulated fluorescence detection and light doubling also allows for direct comparison of
fluorescence measurements on widely differing samples. In a true, dark adapted sample (qp =
1 and em = 0), the modulated measuring beam alone provides an accurate measure of Fo.
Superimposing a saturating light pulse to transiently reduce QA defines the Fmax level (qp =
0, em = 0; Figure 8). These fluorescence levels provide limits between which all other
signals can be normalized (relative fluorescence yield with Fo = 0 and Fmax = 1), permitting
direct comparison of steady-state fluorescence levels and quenching parameters between
samples. The disadvantage of the modulated fluorescence technique is its cost (compared to
the single source technique). Several commercial instruments are available and are seeing
widespread application in higher plants, but in general they lack sensitivity for work with
dilute algal samples. With care, they will work on samples containing 1 Ilg chI a/ml, and
improvements in design could produce another factor of 10 increase in sensitivity.
modulated excitation has two advantages. First, its intensity may be kept low enough that the
measuring source alone produces no actinic effects (such that the modulated source alone
measures only Fo in a dark adapted sample). Use of a lock-in amplifier in the detection
circuitry provides for sufficient signal to noise even at the low excitation intensity. Second,
the modulated fluorescence signal is essentially independent of any direct effects of all nonmodulated sources illuminating the sample. This means that an instantaneous change in the
intensity of a non-modulated source will have no affect on the instantaneous fluorescence
yield. However, the non-modulated sources will have an indirect, actinic effect by altering
the state of the photochemical reactions in the sample. Thus the modulated fluorescence signal
measures a true, relative fluorescence yield. The modulated fluorescence technique and its
advantages are described in detail by Schreiber (1983, 1986).
An additional advantage of the modulated fluorescence measurements is that it provides for
a non-destructive technique for separating photochemical and non-photochemical fluorescence
quenching (Schreiber et aI., 1986). In this procedure, brief (0.1-2 s) illumination with high
intensity light is used to induce a transient, complete reduction of Ch, bringing qp to O. Any
fluorescence quenching remaining when qp = 0 is by definition em (Figure 8). This technique
of "light doubling" was introduced by Bradbury and Baker (1984). The combination of
modulated fluorescence detection and light doubling also allows for direct comparison of
fluorescence measurements on widely differing samples. In a true, dark adapted sample (qp =
1 and em = 0), the modulated measuring beam alone provides an accurate measure of Fo.
Superimposing a saturating light pulse to transiently reduce QA defines the Fmax level (qp =
0, em = 0; Figure 8). These fluorescence levels provide limits between which all other
signals can be normalized (relative fluorescence yield with Fo = 0 and Fmax = 1), permitting
direct comparison of steady-state fluorescence levels and quenching parameters between
samples. The disadvantage of the modulated fluorescence technique is its cost (compared to
the single source technique). Several commercial instruments are available and are seeing
widespread application in higher plants, but in general they lack sensitivity for work with
dilute algal samples. With care, they will work on samples containing 1 Ilg chI a/ml, and
improvements in design could produce another factor of 10 increase in sensitivity.