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Photoacoustic Spectroscopy 9

help to measure photosynthetic oxygen evolution, and the
fluorescence technique can be used to monitor the pho-
tochemistry of photosystem II. Unlike these techniques,
PAS can be used to measure photosynthetic oxygen evo-
lution, and to determine the photochemistry of photosys-
tems I and II. During the past two decades, this technique
has been applied to answer several questions of intact leaf
photochemistry.
Several reviews have been published on the application
of PAS to the photosynthesis of green plants. Using PAS,
certain aspects of intact leaf photosynthesis, which can-
not be understood by other techniques, have been stud-
ied. The study of photosynthetic molecular mechanism
and structure is probably the most popular application of
FIGURE 7A Photoacoustic spectra recorded at room tempera-
PAS.
ture with bovine amelanotic pigment epithelium.
a. Photoacoustic signal generation in green
leaves. Inalltheabovementionedapplications,wehave
been describing that the thermal deactivation of excited
molecules from the sample alone contributes to the PA
signal. However, in green leaves, besides the thermal de-
activation of pigments, the product of the modulated pho-
tochemistry such as photosynthetic oxygen evolution also
contributes to the signal depending on the modulation fre-
quency. In plant cells, chloroplasts are the sites of pho-
tosynthesis. These organelles are suspended in the cyto-
plasm of the cell. Oxygen molecules generated during the
water oxidation phenomenon of primary photochemical
reactions have to travel to the surface of the cell in order
to contribute to the acoustic signal. Thus, the photoacous-
tic signal from leaves is a composite of modulated heat
FIGURE 7B Photoacoustic spectra measured for the melanotic
epithelium. and oxygen emissions depending on the modulation fre-
quency. At low modulation frequency (<200 Hz), both
heat and oxygen contribute to the signal, and at higher
modulation frequencies (>200 Hz), the oxygen signal is
influenced by scattering particles and gas bubbles. PAS
dampened and only heat emission contributes to the sig-
has proved to be very useful in the cases of very low and
nal. The oxygen signal can be dampened by infiltrating
high number of cells; at these concentrations, the trans- the leaves with water; and heat emission alone can also be
mission was found to show pronounced deviation from
measured even at low modulation frequencies.
the true number of cells.
The heat and oxygen emissions can be separated vec-
torially in the presence of a saturated strong background
light using a lock-in amplifier in the two-phase mode by
2. Photosynthesis
recording the signal in the in-phase and quadrature chan-
Another important field where PAS has been successfully nels. The saturated background light closes the reaction
employed is green plant photosynthesis. Although there centers, and all the absorbed modulated measuring light is
are many techniques available to study photosynthesis, not released as heat without contributing to modulated photo-
many are applicable to the comprehensive investigation chemistry. In the presence of background light, the signal
of photosynthesis of intact plants. Even among the tech- in one channel (quadrature) can be adjusted to zero by
niques that measure the intact leaf photosynthesis, their adjusting the phase angle. Upon switching off the back-
applications are limited to specific photosynthetic param- ground light, the modulated measuring light contributes to
eters. For example, an infrared gas analyzer can be used the photochemistry and the oxygen signal can be seen in
only to measure the carbon dioxide uptake during photo- the quadrature channel (Fig. 8). The amplitude of the oxy-
synthesis. Similarly, the oxygen electrode technique can gen signal (A ox ) can be evaluated according to the standard