Fluorescence Induction and Relaxation (FIRe) Technique and Instrumentation for
Monitoring Photosynthetic Processes and Primary Production in Aquatic Ecosystems
Maxim Y. Gorbunov1,2 and Paul G. Falkowski2
1)
2)
Satlantic Inc., Richmond Terminal, Pier 9, 3481 North Marginal Road, Halifax, NS, B3K 5X8, Canada
Institute of Marine and Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, New Jersey 08901, USA
Abstract
Over the last decade, the Fast Repetition Rate Fluorometry
(FRRF) provided tremendous insight into the factors controlling
primary production in the ocean. The use of FRRF became an
integral part of many biological oceanographic programs, but
its broader use is limited by the complexity and high cost of the
available instrumentation. We have designed and built a new
instrument to measure photosynthetic characteristics in
phytoplankton and benthic organisms, such as macrophytes
and corals. The Fluorescence Induction and Relaxation (FIRe)
technique and realized instrumentation are based on similar
biophysical principles as the FRRF. However, the electronic
circuitries and measurement protocols have been simplified
and the optical design has been improved, thus greatly
increasing the sensitivity and reliability and reducing the
production cost. The FIRe technique relies on active
stimulation and highly resolved detection of the induction and
subsequent relaxation of chlorophyll fluorescence yields. A
multicolor excitation source has been implemented to
accommodate efficient excitation of diverse functional groups
within phytoplankton communities. Analysis of fluorescence
induction on microsecond time scales results in the minimum
and maximum fluorescence yields, the quantum efficiency of
photochemistry in PSII, the functional absorption cross-section
of PSII, and the energy transfer between PSII units. The
recorded relaxation kinetics of fluorescence yields reflects the
rates of electron transport on the acceptor side of PSII and
between PSII and PSI. The photosynthetic electron transport
rates as a function of irradiance, together with coefficients of
photochemical and non-photochemical quenching are
measured using an incorporated source of background light. A
benchtop version of the FIRe System is used for
measurements on phytoplankton samples, macrophytes, or
higher plants. The compact design, low power consumption,
and network capability of the submersible version of the FIRe
System make it a robust sensor for long-term monitoring
programs in coastal zones and the open ocean.
The FIRe technique provides a
comprehensive suite of
photosynthetic characteristics:
Fluorescence Induction and
Relaxation profile
The default measurement protocol consists of four
phases: (1) A strong short pulse of 100 ms duration
(called Single Turnover Flash, STF) is applied to
cumulatively saturate PSII and measure the fluorescence
induction from Fo to Fm(STF). (2) Weak modulated light
is applied to record the relaxation kinetics of fluorescence
yield on the time scale of 500 ms. (3) A strong long pulse
of 50 ms duration (called Multiple Turnover Flash, MTF)
is applied to saturate PSII and the PQ pool. (4) Weak
modulated light is applied to record the kinetics of the PQ
pool re-oxidation the time scale of 1s.
Phase 1 provides Fo, Fm, Fv/Fm(STF), sPSII, p; Phase 2
– time constants for the electron transport on the acceptor
side of PSII (re-oxidation of Qa acceptor); Phase 3 Fm(MTF) & Fv/Fm(MTF); Phase 4 – time constant for the
electron transport between PSII and PSI (re-oxidation of
the PQ pool).
Improved assessment of Chla concentration from
variable fluorescence
Fo, F m
Minimum and maximum yields of Chl-a
fluorescence measured in a dark-adapted sate.
Relationship between the minimum
fluorescence, Fo, and Chl-a concentration.
Fv
Variable fluorescence (= F m-Fo)
Fo is the fluorescence yield measured by most of
conventional fluorometers. Fo per unit Chl-a
varies by a factor of ~3-4, depending on the
functional state of the photosynthetic apparatus.
Fv/F m
Maximum quantum yield of photochemistry in
PSII, measured in a dark-adapted state.
s PSII
Functional absorption cross section of PSII.
p
‘Connectivity factor’, defining the exciton energy
transfer between individual photosynthetic units.
Quantum yield of photochemistry in PSII,
measured under ambient light.
Fv’/F m’
Quantum efficiency of photochemistry in open
reaction centers of PSII, measured in a lightadapted state (=(F m’-Fo’)/F m’).
qp
Coefficients of photochemical quenching (=
(F m’-F’)/(F m’-Fo’)),
qN
Coefficients of non-photochemical quenching,
i.e. (F m-F m’)/(F m-F o).
tQa
Time constant for the electron transport on the
acceptor side of PSII (the time of Qa reoxidation)
tPQ
Time constant for the electron transport
between PSII and PSI (the time of the PQ pool
re-oxidation)
Signatures of Iron
Limitation
•
•
[Chl-a] FIRe = C(Fv/F m, s PSII) * F o
C - fluorescence per unit Chl-a calculated from
photosynthetic characteristics.
•
Increase in Fv/F m;
Decrease in fluorescence per
unit chlorophyll;
Increase in the rate of Qa reoxidation (i.e., the rate at which
light-induced electrons can be
used in photosynthetic
reactions),
Decrease in the functional
absorption cross section of PSII,
s PSII.
Project Objectives:
• Develop advanced optical techniques for assessment of the
viability and health of coral reef communities with the capabilities of
selective identification of natural and anthropogenic stresses.
Fluorescent
Diagnostics of Thermal
Stress in Coral
• Collect a library of baseline data on physiological, biophysical, biooptical and genetic diversity of coral reef communities in three major
geographic areas.
• a characteristic decrease in the
quantum yield of photochemistry in PSII
(Fv/Fv) under both stresses
• Extremely sensitive (down to 0.02 mg/m3 of Chl-a);
•· Wide dynamic range of the fluorescence signals (four
orders of magnitude);
•· Comprehensive suite of fluorescent and photosynthetic
characteristics;
•· User-friendly and flexible protocols for measurements
and data analysis.
Bench-top versions of the FIRe
Fluorometer System
Measurements are conducted on phytoplankton samples
(in a regular or flow-through cuvettes), leaves, or in lab
aquaria (using a water-tight fiber extension, shown on
the left). The FIRe Fluorometer System manufactured by
Satlantic Inc. is shown on the right (see Exhibit 401 for
detail).
FIRe profiles measured in phytoplankton samples
in the Sargasso Sea (ultra-oligotrophic waters).
Chl-a concentrations for each sample are shown
on the right.
S outhern Ocean SOFEX Experiment, North patch
Analysis of the Physiological State of DoD Coral Reef Communities using
Advanced Fluorescence Techniques
• Develop FIRe Fluorosensors for permanent underwater monitoring
stations and Remote Operated Vehicles.
FIRe System Features
+Fe
When phytoplankton is iron limited,
iron enrichment leads to :
•
Incorporation of photosynthetic parameters into
the model increases dramatically the precision of
fluorescence-based estimates of Chl-a
concentration.
Fo’, F’, F m’ Minimum, steady-state, and maximum yields of
chlorophyll-a fluorescence measured under
ambient light.
DF’/F m’
Biophysical Assessment of Nutrient Limitation in
Phytoplankton by Using Variable Fluorescence
Thermal Stress and Coral Bleaching:
Elucidated Mechanisms and Bio-Optical
Signatures
• but the thermal stress is accompanied
by a striking increase in the time
constant of Qa reoxidation (tQa).
• thermal sensitivity is determined by the membrane lipid
composition of symbiotic algae,
• the thermal stress can be
distinguished from photoinhibition by
using the FIRe technique
• thermal stress starts with disruption of thylakoid membranes
followed by damage to the photosynthetic machinery (PSII);
• accumulation of Reactive Oxygen Species (ROS) produced by the
stresses algae triggers coral death (via Programmed Cell Death);
• the stress development is accompanied by unique fluorescent and
photosynthetic signatures and can be readily diagnosed by the FIRe
technique, even at early stages. (see Tchernov et al., Proc. Natl.
Acad. Sci. USA, in press for detail)
The impact of thermal and high light stresses on
photosynthetic characteristics in the coral
Montastraea anularis
Assessing the effect of eddy induced
nutrient pumping on phytoplankton
photosynthesis the Sargasso Sea
Acknowledgements:
Vertical profiles of fluorescence (a), chlorophyll concentration
(b), the quantum yield of photochemistry in PSII (c), and the
functional absorption cross-section on PSII (c) measured at two
stations with deep (open dots) and shallow (closed dots)
nitrocline.
This work was funded by the U.S. Department of Defense, through the Strategic
Environmental Research and Development Program (SERDP), and NSF. We
thank Denis Klimov, Dan Tchernov, Zbignew Kolber, Christopher M. Graziul, Tony
Quigg, Kevin Wyman, Tomas Bibby, Matt Bochoff, Geoff MacIntyre, Scott McLean,
and Marlon Lewis for assistance and discussion.
See S2D-4 - Abstract number: 41 by Bibby et al. for detail.