CHAPTER 12 MEASUREMENTS WITH THE MINI-PAM
12.2 The PAM measuring principle
With conventional chlorophyll fluorometers, the same light is
used for driving photosynthesis and for exciting fluorescence.
Separation of fluorescence from stray excitation light then is
achieved by appropriate combinations of optical filters (e.g.
excitation by blue light and protection of the detector by a red filter,
which only passes the red fluorescence). Such conventional
fluorometers are of rather limited use for ecophysiological research,
as their function is severely disturbed by ambient daylight. In order
to distinguish between fluorescence and other types of light reaching
the photodetector, fluorescence excitation can be 'modulated': When
a special 'measuring beam' is rapidly switched on/off, the
fluorescence signal follows this on/off pattern and with the help of
suitable electronic devices the resulting modulated signal can be
separated. Standard devices for this purpose are lock-in amplifiers
which tolerate background signals several hundred times larger than
the fluorescence signal. For the extreme requirements of chlorophyll
fluorescence quenching analysis by the so-called saturation pulse
method (see 12.1), a new modulation principle was developed which
tolerates a ratio of 1:10
6
between fluorescence and background
signal. This measuring principle is patented (DE 35 18 527) and
licensed exclusively to the Heinz Walz GmbH.
The pulse-amplitude-modulation (PAM) principle displays the
following essential features (see also Fig. 11):
Fluorescence is excited by very brief but strong light pulses from
light-emitting diodes. With the MINI-PAM, these pulses are 3 µs
long and repeated at a frequency of 600 or 20000 Hz. The LED light
passes a short-pass filter (λ<670 nm) and the photodetector is
protected by a long-pass filter (λ>700 nm) and a heat reflecting filter.
A highly selective pulse amplification system ignores all signals
except the fluorescence excited during the 3 µs measuring pulses.
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