Box 3.1 Use of Chlorophyll Fluorescence to
Measure Photosynthetic Activity
(^) Light energy absorbed by chlorophyll can be directed in three ways: energizing photosynthesis
(photochemistry); dissipation as heat; or re-emission as fluorescence. Photochemistry includes the
activity of PSI, PSII and the assimilation of carbon. The spectrum of emitted fluorescence has a peak
at a longer wavelength than the absorbed light. To measure emitted fluorescence, a light source is
switched on and off at high frequency and the detector is tuned to detect only fluorescence excited by
the stimulating light.
(^) Pulse-amplitude-modulated (PAM) fluorometers and fast-repetition-rate fluorometers (FRRF) are
both used to measure the photosynthetic activity of phytoplankton and cyanobacteria. We provide a
simplified explanation of PAM fluorometry here (Mackey et al. 2008) and refer the reader to Kolber
et al. (1998) and Suggett et al. (2009) for details of the FRRF technique. Variable fluorescence (Fv
from Box Fig. 3.1.1) is the maximum fluorescence to bright flashes of dark-adapted cells minus the
fluorescence to standardized dim flashes, Fv = (Fm − Fo). Fv provides an estimate of the maximum
potential rate of electron transport through PSII for dark-adapted cells. The maximum potential
relative photosynthetic efficiency is proportional to Fv. The relative fluorescence decreases with
continuing exposure to light and reaches an asymptote (Fs) after about 5 minutes. Fluorescence
emission (Fs) is smaller because some of the electron acceptors are reduced and no longer able to
accept electrons. The fluorescence response of light-adapted cells to a saturating flash is Fm′. The
actual relative photosynthetic efficiency of PSII in the light-adapted state is (Fm′ − Fs)/Fm′, a ratio
called ΦPSII. Multiplying ΦPSII by the intensity of photosynthetically effective light, IA, provides a
measure of the PSII electron transport rate at that intensity. Thus, ΦPSII · IA is an estimate of gross
photosynthetic oxygen production. See Mackey et al. (2008) for thorough derivation of those
relationships.
Box Fig. 3.1.1 The bar along the top shows periods of dark (black) and actinic
light (white). Minimum fluorescence values (Fo and Fo′) are determined using a
low-intensity measuring light prior to delivery of the saturating light pulses.
Arrows indicate the timing of saturating (3000 μmol quanta m−2−s−1) actinic
light pulses of 0.8 s duration.
(^) (After Mackey et al. 2008.)
(^) PAM fluorometers generate data for Fo, Fm, Fs, Fo′, and Fm′ by the flashing and ‘actinic’ interval
shown in Box Fig. 3.1.1. FRRF systems work on different principles, but are fast enough to generate
roughly similar estimates as water column profiles. Both allow much higher spatial and temporal