Abstract

1. Fm Fo Fv Fv/Fm • Theory of Fluorescence Lifetime and Quantum Yield • Why Measure Fluorescence Lifetimes? • Fluorescence is a delayed emission which is described by the exponential decay function and is characterized by the lifetime of fluorescence. • The fluorescence lifetime is directly proportional to the quantum yield of fluorescence (ff): •  = f xt0 • where is the observed lifetime of the excited singlet state of the molecule;  is its natural lifetime. • The lifetime of fluorescence from isolated Chl-a molecules (e.g., in either solution) is 5.1 ns and the quantum yield is 32%. The natural lifetime of Chl-a fluorescence is 15 ns. • The lifetimes of Chl-a fluorescence in living cells are considerably shorter due to efficient photochemical use of absorbed energy. • In vivo Chl-a fluorescence lifetimes vary between 0.3 and 1.5 ns, depending on the physiological state of the cells. Fluorescence Lifetime Measurements (Closed RCs, dark) Strong Light (NPQ effect) Open RCs Picosecond kinetics of fluorescence decay in Chlorella. Fo - dark-adapted cells with open reaction centers (the quantum yield of photosynthesis is maximum). Fm - dark-adapted cells with closed centers (photosynthesis is zero). Fm’ – under high light. The dramatic reduction in the fluorescence lifetime under high light is due to non-phochemical quenching (NPQ). Time Correlated Single Photon Counting (TCSPC) System Application of Lifetime Analyses in the Upper Ocean to the Interpretation of Satellite-Based, Solar Induced Chlorophyll Fluorescence Signals Maxim Y. Gorbunov and Paul G. Falkowski Institute of Marine & Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, NJ 08901 Development of Sea-Going Instrumental Package to Measure Fluorescence Yields, Lifetimes, and Photosynthetic Characteristics of Phytoplankton Abstract The distribution of phytoplankton biomass and primary production in the ocean can be studied from satellite platforms (MODIS and MERIS) using solar induced fluorescence (SIF) from chlorophyll a (ca. 675 nm band). Although the SIF algorithm is potentially most advantageous in Case 2 waters, where DOM and suspended sediments interfere with chlorophyll algorithms based on blue-green water leaving radiances, the application of SIF to understanding either phytoplankton physiology or carbon fixation on a global scale has not been achieved. Chlorophyll fluorescence from phytoplankton is strongly affected by physiological state and taxonomic composition. Basic photosynthesis research over the last decade tremendously improved our understanding of the physiological mechanisms that control the quantum yields of chlorophyll fluorescence, however, the contributions of these mechanisms to the observed variability of SIF in the ocean remain largely unknown. Although the variability in SIF yields is correlated with environmental forcing, the mechanisms responsible for this variability are not known due to very limited field studies of related processes. In this start-up project, we develop an instrumental package to directly measure fluorescence lifetimes and yields in the ocean and to study the physiological mechanisms of variability in the quantum yield of SIF with an overarching goal to develop improved models and algorithms for retrieving phytoplankton biomass, physiological status, and the rates of primary production. The quantum yield of chlorophyll fluorescence is directly derived from Time Correlated Single Photon Counting measurements of fluorescence lifetimes and is integrated with the FIRe variable fluorescence technique. We envision that this research project will provide the scientific background for the interpretation of solar induced fluorescence signals in the ocean, help to improve MODIS biomass algorithms, and provide crucial physiological information needed for better estimates of primary production from remote sensing of the ocean color. Variable Fluorescence Measurements: Assessment of Biomass and Physiological Status of Phytoplankton Single Measurement Time Series Fluorescence Induction and Relaxation (FIRe) System Laboratory Studies • Objectives • Develop a sea-going instrumental package designed to directly measure fluorescence lifetimes and yields in the ocean. The package integrates Time Correlated Single Photon Counting measurements of fluorescence lifetimes with variable fluorescence technique; • Establish the physiological and biophysical mechanisms of variability in the quantum yield of SIF in the ocean with an overarching goal to develop improved models and algorithms for retrieving phytoplankton biomass, physiological status, and the rates of primary production. • Background and Problem Statement • Solar Induced Fluorescence (SIF) from chlorophyll a (Chl-a) is detected as a red peak (ca. 675 nm) in spectra of water-leaving radiance. • SIF is the only signal emitted from the ocean and detectable from space that can be unambiguously ascribed to phytoplankton. • The SIF algorithm is potentially most advantageous in Case 2 waters, where DOM and suspended sediments interfere with chlorophyll algorithms based on blue-green ratios (Gower et al, 1999; Hoge et al, 2003; Siegel et al, 2005; Cullen, 2007; Huot et al, 2007). • While the huge variability (ca. 10x) in SIF yield in the ocean is often correlated with environmental forcing (Letelier et al, 1996; Morrison 2003; Huot et al, 2005), the mechanisms and interpretation of this relationship remain to be elucidated. Taxonomic variability in the capacity of non-photo-chemical quenching (NPQ). Brown algae (diatom and dinoflagellates) exhibit the strongest NPQ due to the efficient light-induced xanthophyll cycle. In contrast, red algae and cyanobacteria did not evolve xanthophyll cycle mechanism and show relatively weak NPQ that is driven by energy-dependent quenching (qE) induced by the pH gradient across the thylakoid membranes. The Irradiance Dependence of Chlorophyll Fluorescence Yields Fo and Fm are the minimum (open reaction centers) and maximum (closed centers) fluorescence yields measured in darkness. Fo’ and Fm’ are the minimum and maximum fluorescence yields measured in light-adapted cells. F’ is the actual fluorescence yield measured under ambient light that corresponds to the yield of remotely sensed Solar Induced Fluorescence. PQ and NPQ are photochemical quenching (i.e., phytosynthetic use) and non-photochemical quenching (thermal dissipation), respectively. While fluorescence yields under low light are primarily controlled by the photosynthetic efficiency, SIF yields (under high light) are controlled by the magnitude of non-photochemical quenching (NPQ). The measurements were made in a marine diatom, Pheoductylum tr.,by using a FIRe fluorometer (Gorbunov and Falkowski 2005). References Cullen, J. J., A. M. Ciotti, R. F. Davis and P. J. Neale. (1997) In: Ocean Optics XIII, S.G. Ackleson and R. Frouin, eds. Proc. SPIE 2963: 272-277. Gorbunov MY, and Falkowski PG (2005) In: “Photosynthesis: Fundamental Aspects to Global Perspectives” (Eds: A. van der Est and D. Bruce), Allen Press, V.2, pp. 1029-1031. Gower, J.F.R., R. Doerffer, and G.A. Borstad. (1999) - Int. J. Remote Sens. 20: 1771–1786. Hoge FE, Lyon PE, Swift RN, Yungel JK, MR Abbott, RM Letelier, and WE Esaias (2003) - Applied Optics, 42 (15): 2767-2771. Huot Y, CA Brown, and JJ Cullen (2005) - Limnol. Oceanogr.: Methods v.3. Huot, Y., C. A. Brown, and J. J. Cullen. (2007) - Journal of Geophysical Research, Vol. 112, C06013, doi:10.1029/2006JC003794 Letelier, R.M., and M.R. Abbott (1996) - Rem. Sensing Environ., 58, 215-223, 1996. Morrison JR (2003) – Limnol. Oceanogr., 48(2): 618-631. Siegel, D. A., S. Maritorena, N. B. Nelson, M. J. Behrenfeld, and C. R. McClain (2005) - Geophys. Res. Lett., 32, L20605, doi:10.1029/2005GL024310. Effect of photoacclimation on the capacity of NPQ. The capacity of NPQ decreases dramatically with decrease in growth irradiance. Growth irradiance: HL = 500, ML = 100, LL = 20 mole quanta m-2 s-1. Measurements are made in the diatom, Dilytum br. This work is funded by NASA (NNX08AC24G, 2008-2010).