Calibration of an in-water multi-excitation fluorometer for the measurement of phytoplankton chlorophyll-a fluorescence quantum yield

Abstract

A multi-excitation fluorometer (MFL, JFE Advantech Co., Ltd.), originally designed to discriminate between phytoplankton species present within a population, has been redirected for use in fluorescence quantum yield (FQY) determination. While this calibration for apparent FQY requires no modification of the MFL, it is necessary to have an independent measurement of the spectral absorption coefficient of the subject fluid. Two different approaches to calibration were implemented. The primary method made use of reference fluorescent dye solutions of known quantum yield. The second method made use of acrylic fluorescent plaques and films. The two methods yielded consistent results, except in the 570 and 590 nm LED channels of the MFL. Application of the MFL in FQY determination is illustrated with an in situ Southern Ocean sample.

Spatial and temporal drivers of fluorescence quantum yield variability in the Southern Ocean

“…This is likely due to the lack of dedicated instrumentation that can simultaneously measure absorption together with the incident and returning light levels required to determine in situ Φ F . In this study, separate measurements of phytoplankton absorption and wavelength‐specific fluorescence were measured, before deriving wavelength‐specific Φ F ( λ ) using the calibration constants from Griffith et al (2018). The variability of Φ F ( λ ) was then investigated against potential in situ drivers across a range of latitudes and two separate seasons in the Southern Ocean, the austral winter of 2012 and summer of 2013.…”

“…Φ F ( λ ) were subsequently derived for each MFL excitation wavelength using wavelength‐specific fluorescence, spectrally averaged a ph (see “Light history” section) and the MFL calibration coefficients from Griffith et al (2018) as described below in Eq. 1: where R ( λ ) is the MFL signal response at each wavelength, k ( λ ) is the calibration constant from the ATTO 490LS dye ( see Griffith et al 2018 for more details), aph ( λ ) is the wavelength specific phytoplankton absorption normalized to the spectra of the MFL‐LED ( λ ), and P c is the MFL‐relative partial quantum yield factor comprised of the mean values of Chl a in ether and methanol ( P c = 0.851). As the MFL filters out part of the dye emission it is necessary to calculate the relative partial quantum yield, which lies between 0 and 1 and takes into account the spectral response of the MFL detector channel.…”

“…The aim of this study was to deploy the MFL in the field to derive absolute Φ F ( λ ) measurements in the Atlantic Southern Ocean in austral winter (2012) and summer (2013/2014) to address the hypothesis that seasonality would be the dominant driver in Φ F ( λ ). The calibration factors derived by Griffith et al (2018) were used together with underway (UW) real‐time measurements of MFL‐fluorescence and phytoplankton specific absorption measurements to calculate absolute Φ F ( λ ) values. Spectral analysis was performed on all derived Φ F ( λ ) such that potential relationships between Φ F ( λ ) and possible drivers of variability could be established.…”

“…The derivation of Φ F requires, besides accurate quantification of the fluorescence signal, knowledge of the spectral absorption capacity of phytoplankton and a fully characterized light field. Field studies of Φ F typically involve SICF measurements (Letelier et al 1997;Maritorena et al 2000;Morrison 2003;Schallenberg et al 2008) or proxies derived from active fluorometry (Browning et al 2014;Lin et al 2016); however, the use of multiexcitation fluorometers in the derivation of Φ F has been proposed (Ostrowska 2012;Griffith et al 2018). Multiexcitation fluorometers typically exploit fluorescence excitation spectra to define phytoplankton taxonomic groups based on signature accessory pigment composition (Sosik and Mitchell 1995;Johnsen et al 1997), which is used to discriminate between phytoplankton species in mixed assemblages (Cowles et al 1993;Beutler et al 1998Beutler et al , 2002Beutler et al , 2003.…”

“…Although they did not radiometrically characterize the excitation light-emitting diodes (LEDs) nor the emission detector of the instrument, they were still able to infer apparent, wavelength-specific Φ F values. More recently, Griffith et al (2018) employed a MFL to derive fully characterized absolute measurements of wavelength-specific Φ F . The advantages of wavelength-specific Φ F over measurements centered around the peak absorption of Chl a is that it allows us to determine the importance of pigments that span a large range of the absorption spectra, which includes both photoprotective and photosynthetic carotenoids, under different environmental conditions.…”