CHAPTER 3 PAM FLUOROMETRY & SAT PULSE ANALYSIS
fluorometer MONI-PAM, and imaging fluorometers (IMAGING-PAM
M series) have been successfully used all over the world.
PAM fluorometers, as many conventional fluorometers, use filter com-
binations which prevent the fluorometer’s excitation radiation from
reaching the photodetector. Different from conventional devices, the
PAM fluorometers measure only the fluorescence resulting from an
evenly pulsed measuring beam: the fluorescence caused by continuous
radiation is not measured. This permits the use of continuous radiation
to manipulate the fluorescence yield in photosynthetic membranes and
to carry out the so-called saturating pulse analyses.
Subsequently, the Pulse-Amplitude-Modulated fluorescence signal and
the saturating pulse analysis will be briefly explained.
3.1
Pulse-Amplitude-Modulated Fluorescence
In panel A of Figure 3.1, an excitation intensity regime is depicted
which consists of two separate 80 s intervals of actinic illumination of
different intensities. Always, a pulsed light source with constant pulse
height but rather low intensity is active. The pulsed radiation corre-
sponds to the measuring light of a PAM fluorometer.
If the latter light regime excites fluorescence from a JUNIOR-PAM
fluorescence standard, the signal obtained with a conventional fluoro-
meter is simply proportional to the excitation intensity (Fig. 3.1 B
“standard”). If the conventional instrument records fluorescence from a
leaf, typical “Kautsky-type” induction curves appear during actinic il-
lumination (Fig. 3.1 B “leaf”). For the same actinic light intensity, these
variations are determined by changes in the fluorescence yield. In addi-
tion, the intensity of actinic light controls the fluorescence signal as de-
monstrated by the upward shifted induction kinetics in the presence of
the higher actinic intensity compared to the kinetics obtained with lower
actinic light. Hence, conventional fluorometers respond to both, varia-
tions in fluorescence quantum yield and excitation light intensity.
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