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detected (the measuring or excitation function), and to drive the photosynthetic reactions or
to poise the sample in some known physiological state (the actinic function). Techniques for
measuring chI fluorescence can be divided into three categories depending on the type of light
source(s) used to provide these functions: (1) a single, continuous light source which acts both
as a measuring and actinic source, (2) two or more sources using one low intensity,
modulated source to excite fluorescence and separate, non-modulated source(s) to provide
actinic illumination, (3) pulsed light sources. Each of these general techniques has inherent
benefits and disadvantages which will be discussed briefly. In general, each of these
techniques can be applied to dilute suspensions of phytoplankton or to samples in which the
cells have been concentrated on a filter substrate. The minimum concentration of chI required
for analysis is highly instrument-dependent. Any manipulation of samples prior to
measurement (concentration, filtration or storage) must be considered as potentially altering
the sample from its in situ characteristics.
Instruments using a single light source as both exciting and actinic illumination are most
commonly used for measurement of fluorescence induction. In general, the sample is dark
adapted to bring qp = 1 and em = O. The sample is then suddenly exposed to continuous
illumination (usually using a shutter) and the time-dependent changes in fluorescence emission
are monitored (Figure 7). Illumination both excites the fluorescence and drives the induction
process. Estimation of the components of fluorescence quenching can be accomplished in this
system by taking advantage of the different rates at which the various quenching processes
relax when electron transport is inhibited by the rapid (1 s) addition of DCMU (Walters and
Horton, 1990). The single source technique has the benefits of being inexpensive, requiring
relatively unsophisticated apparatus and has been widely used in a number of different
applications. The system suffers from considerable inflexibility in measurements, primarily
the inability to provide internal standards (for normalizing fluorescence between samples and
to quantify the components of fluorescence quenching) without poisoning the sample with
DCMU. In addition, any change in source intensity will be detected as changes in
fluorescence emission independent of the quenching state of the sample.
Measurement of fluorescence using a modulated excitation source and continuous actinic
source(s) overcomes the experimental limitations in the single source technique. Use of the
detected (the measuring or excitation function), and to drive the photosynthetic reactions or
to poise the sample in some known physiological state (the actinic function). Techniques for
measuring chI fluorescence can be divided into three categories depending on the type of light
source(s) used to provide these functions: (1) a single, continuous light source which acts both
as a measuring and actinic source, (2) two or more sources using one low intensity,
modulated source to excite fluorescence and separate, non-modulated source(s) to provide
actinic illumination, (3) pulsed light sources. Each of these general techniques has inherent
benefits and disadvantages which will be discussed briefly. In general, each of these
techniques can be applied to dilute suspensions of phytoplankton or to samples in which the
cells have been concentrated on a filter substrate. The minimum concentration of chI required
for analysis is highly instrument-dependent. Any manipulation of samples prior to
measurement (concentration, filtration or storage) must be considered as potentially altering
the sample from its in situ characteristics.
Instruments using a single light source as both exciting and actinic illumination are most
commonly used for measurement of fluorescence induction. In general, the sample is dark
adapted to bring qp = 1 and em = O. The sample is then suddenly exposed to continuous
illumination (usually using a shutter) and the time-dependent changes in fluorescence emission
are monitored (Figure 7). Illumination both excites the fluorescence and drives the induction
process. Estimation of the components of fluorescence quenching can be accomplished in this
system by taking advantage of the different rates at which the various quenching processes
relax when electron transport is inhibited by the rapid (1 s) addition of DCMU (Walters and
Horton, 1990). The single source technique has the benefits of being inexpensive, requiring
relatively unsophisticated apparatus and has been widely used in a number of different
applications. The system suffers from considerable inflexibility in measurements, primarily
the inability to provide internal standards (for normalizing fluorescence between samples and
to quantify the components of fluorescence quenching) without poisoning the sample with
DCMU. In addition, any change in source intensity will be detected as changes in
fluorescence emission independent of the quenching state of the sample.
Measurement of fluorescence using a modulated excitation source and continuous actinic
source(s) overcomes the experimental limitations in the single source technique. Use of the