Using Polarization to Increase Contrast of Water OH Raman Scattering Relative to Fluorescence of Dissolved Organic Matter.

Environmental fluorescence measurements sometimes use water Raman scattering as an internal standard to compensate for path length, lensing effects, and turbidity. Fluorescent dissolved organic matter (FDOM) in water may interfere strongly with the measurement of this reference. However, fluorescence in fluid solution is largely unpolarized, while the OH stretching Raman band of water is always strongly polarized.

Variability in spectral absorption within cryptophyte phycobiliprotein types.

Cryptophytes are known to vary widely in coloration among species. These differences in color arise primarily from the presence of phycobiliprotein accessory pigments. There are nine defined cryptophyte phycobiliprotein (Cr-PBP) types, named for their wavelength of maximal absorbance.

A three-genome ultraconserved element phylogeny of cryptophytes.

Cryptophytes are single celled protists found in all aquatic environments. They are composed of a heterotrophic genus, Goniomonas, and a largely autotrophic group comprising many genera. Cryptophytes evolved through secondary endosymbiosis between a host eukaryotic heterotroph and a symbiont red alga.

Consequences of light spectra for pigment composition and gene expression in the cryptophyte Rhodomonas salina.

Algae with a more diverse suite of pigments can, in principle, exploit a broader swath of the light spectrum through chromatic acclimation, the ability to maximize light capture via plasticity of pigment composition. We grew Rhodomonas salina in wide-spectrum, red, green, and blue environments and measured how pigment composition differed. We also measured expression of key light-capture and photosynthesis-related genes and performed a transcriptome-wide expression analysis.

Filter Fluorometer Calibration Without the Fluorometer.

We recently described a lightweight, low-power, waterproof filter fluorometer using a 180° backscatter geometry for chlorophyll-a (chl-) detection. Before it was constructed it was modeled to ensure it would have satisfactory performance. This manuscript repeats the modeling process that allows the calibration slope and detection limit for a fluorescent analyte in water to be estimated from system component performance and conventional spectrofluorometry alone.

Fluorometer Control and Readout Using an Arduino Nano 33 BLE Sense Board.

We describe the control and interfacing of a fluorometer designed for aerial drone-based measurements of chlorophyll- using an Arduino Nano 33 BLE Sense board. This 64 MHz controller board provided suitable resolution and speed for analog-to-digital (ADC) conversion, processed data, handled communications via the Robot Operating System (ROS) and included a variety of built-in sensors that were used to monitor the fluorometer for vibration, acoustic noise, water leaks and overheating. The fluorometer was integrated into a small Uncrewed Aircraft System (sUAS) for automated water sampling through a Raspberry Pi master computer using the ROS.

Chlorophyll Fluorometer for Intelligent Water Sampling by a Small Uncrewed Aircraft System (sUAS).

We describe a waterproof, lightweight (1.3 kg), low-power (∼1.1 W average power) fluorometer operating on 5 V direct current deployed on a small uncrewed aircraft system (sUAS) to measure chlorophyll and used for triggering environmental water sampling by the sUAS.

Photopigment, Absorption, and Growth Responses of Marine Cryptophytes to Varying Spectral Irradiance.

The underwater light field of lakes, estuaries, and oceans may vary greatly in spectral composition. Phytoplankton in these environments must contain pigments that absorb the available colors of light. If spectral quality changes, acclimation to the new spectral environment would confer an ecological advantage in terms of photosynthesis and growth.

Diversification of light capture ability was accompanied by the evolution of phycobiliproteins in cryptophyte algae.

Evolutionary biologists have long sought to identify phenotypic traits whose evolution enhances an organism's performance in its environment. Diversification of traits related to resource acquisition can occur owing to spatial or temporal resource heterogeneity. We examined the ability to capture light in the Cryptophyta, a phylum of single-celled eukaryotic algae with diverse photosynthetic pigments, to better understand how acquisition of an abiotic resource may be associated with diversification.

Light capture and pigment diversity in marine and freshwater cryptophytes.

Phenotypic traits associated with light capture and phylogenetic relationships were characterized in 34 strains of diversely pigmented marine and freshwater cryptophytes. Nuclear SSU and partial LSU rDNA sequence data from 33 of these strains plus an additional 66 strains produced a concatenated rooted maximum likelihood tree that classified the strains into 7 distinct clades. Molecular and phenotypic data together support: (i) the reclassification of Cryptomonas irregularis NIES 698 to the genus Rhodomonas, (ii) revision of phycobiliprotein (PBP) diversity within the genus Hemiselmis to include cryptophyte phycocyanin (Cr-PC) 569, (iii) the inclusion of previously unidentified strain CCMP 2293 into the genus Falcomonas, even though it contains cryptophyte phycoerythrin 545 (Cr-PE 545), and (iv) the inclusion of previously unidentified strain CCMP 3175, which contains Cr-PE 545, in a clade with PC-containing Chroomonas species.

Single-Cell and Bulk Fluorescence Excitation Signatures of Seven Phytoplankton Species During Nitrogen Depletion and Resupply.

Phytoplankton play a vital role as primary producers in aquatic ecosystems. One common approach to classifying phytoplankton is fluorescence excitation spectroscopy, which leverages the variation in types and concentrations of pigments among different phytoplankton taxonomic groups. Here, we used a fluorescence imaging photometer to measure excitation ratios ("signatures") of single cells and bulk cultures of seven differently pigmented phytoplankton species as they progressed from nitrogen N-replete to N-depleted conditions.

Asymmetric Versus Symmetric Filter Wheels and Associated Processing Algorithms: Results from Asynchronous Fluorescence Imaging Photometer Measurements of Phytoplankton.

The use of rotating filter wheels is common in photometric applications. Traditional filter wheel designs typically exhibit a number of filter openings spaced evenly about the circumference of the wheel. In this work we examine a number of shortcomings of this traditional filter design in measurements of phytoplankton fluorescence made with our fluorescence imaging photometer (FIP).

Mechanisms and Pathways of Small-Phytoplankton Export from the Surface Ocean.

Carbon fixation by phytoplankton near the surface and the sinking of this particulate material to deeper waters are key components of the biological carbon pump. The efficiency of the biological pump is influenced by the size and taxonomic composition of the phytoplankton community. Large, heavily ballasted taxa such as diatoms sink quickly and thus efficiently remove fixed carbon from the upper ocean.

Fluorescence Excitation Spectroscopy for Phytoplankton Species Classification Using an All-Pairs Method: Characterization of a System with Unexpectedly Low Rank.

An all-pairs method is used to analyze phytoplankton fluorescence excitation spectra. An initial set of nine phytoplankton species is analyzed in pairwise fashion to select two optical filter sets, and then the two filter sets are used to explore variations among a total of 31 species in a single-cell fluorescence imaging photometer. Results are presented in terms of pair analyses; we report that 411 of the 465 possible pairings of the larger group of 31 species can be distinguished using the initial nine-species-based selection of optical filters.

Differential effects of changes in spectral irradiance on photoacclimation, primary productivity and growth in Rhodomonas salina (Cryptophyceae) and Skeletonema costatum (Bacillariophyceae) in simulated blackwater environments.

The underwater light field in blackwater environments is strongly skewed toward the red end of the electromagnetic spectrum due to blue light absorption by colored dissolved organic matter (CDOM). Exposure of phytoplankton to full spectrum irradiance occurs only when cells are mixed up to the surface. We studied the potential effects of mixing-induced changes in spectral irradiance on photoacclimation, primary productivity and growth in cultures of the cryptophyte Rhodomonas salina and the diatom Skeletonema costatum.

Ecotoxicology of bromoacetic acid on estuarine phytoplankton.

Bromoacetic acid is formed when effluent containing chlorine residuals react with humics in natural waters containing bromide. The objective of this research was to quantify the effects of bromoacetic acid on estuarine phytoplankton as a proxy for ecosystem productivity. Bioassays were used to measure the EC50 for growth in cultured species and natural marine communities.

Focus-independent particle size measurement from streak images: a comparison of multivariate methods.

Our laboratories have recently developed a flow-through imaging photometer to characterize and classify fluorescent particles between 3 and 47 μm in size. The wide aperture of the objective lens (0.7 NA) required for measuring spectral fluorescence of single particles restricts the depth of field, such that a large sample volume results in many particles that are out of focus.

Pigment composition and photoacclimation as keys to the ecological success of Gonyostomum semen (Raphidophyceae, Stramenopiles).

Aquatic habitats are usually structured by light attenuation with depth resulting in different microalgal communities, each one adapted to a certain light regime by their specific pigment composition. Several taxa contain pigments restricted to one phylogenetic group, making them useful as marker pigments in phytoplankton community studies. The nuisance and invasive freshwater microalga Gonyostomum semen (Raphidophyceae) is mainly found in brown water lakes with sharp vertical gradients in light intensity and color.

Taxonomic classification of phytoplankton with multivariate optical computing, part III: demonstration.

We describe the automatic analysis of fluorescence tracks of phytoplankton recorded with a fluorescence imaging photometer. The optical components and construction of the photometer were described in Part I and Part II of this series in this issue. An algorithm first isolates tracks corresponding to a single phytoplankter transit in the nominal focal plane of a flow cell.

Taxonomic classification of phytoplankton with multivariate optical computing, part II: design and experimental protocol of a shipboard fluorescence imaging photometer.

Differential pigmentation between phytoplankton allows use of fluorescence excitation spectroscopy for the discrimination and classification of different taxa. Here, we describe the design and performance of a fluorescence imaging photometer that exploits taxonomic differences for discrimination and classification. The fluorescence imaging photometer works by illuminating individual phytoplankton cells through an asynchronous spinning filter wheel, which produces bar code-like streaks in a fluorescence image.

Taxonomic classification of phytoplankton with multivariate optical computing, part I: design and theoretical performance of multivariate optical elements.

Phytoplankton are single-celled, photosynthetic algae and cyanobacteria found in all aquatic environments. Differential pigmentation between phytoplankton taxa allows use of fluorescence excitation spectroscopy for discrimination and classification. For this work, we applied multivariate optical computing (MOC) to emulate linear discriminant vectors of phytoplankton fluorescence excitation spectra by using a simple filter-fluorometer arrangement.

Estimation of chromophoric dissolved organic matter (CDOM) and photosynthetic activity of estuarine phytoplankton using a multiple-fixed-wavelength spectral fluorometer.

The utility of a multiple-fixed-wavelength spectral fluorometer, the Algae Online Analyser (AOA), as a means of quantifying chromophoric dissolved organic matter (CDOM) and phytoplankton photosynthetic activity was tested using algal cultures and natural communities from North Inlet estuary, South Carolina. Comparisons of AOA measurements of CDOM to those by spectrophotometry showed a significant linear relationship, but increasing amounts of background CDOM resulted in progressively higher over-estimates of chromophyte contributions to a simulated mixed algal community. Estimates of photosynthetic activity by the AOA at low irradiance (≈ 80 μmol quanta m(-2) s(-1)) agreed well with analogous values from the literature for the chlorophyte, Dunaliella tertiolecta, but were substantially lower than previous measurements of the maximum quantum efficiency of photosystem II (F(v)/F(m)) in Thalassiosira weissflogii (a diatom) and Rhodomonas salina (a cryptophyte).

Spectral fluorometric characterization of phytoplankton community composition using the Algae Online Analyser.

The utility of a multiple-fixed-wavelength spectral fluorometer, the Algae Online Analyser (AOA), as a means of quantifying phytoplankton biomass and community composition was tested using natural communities from two southeastern United States estuaries, North Inlet, South Carolina, and the Neuse River Estuary, North Carolina. Estimates of biomass (as chlorophyll a) were correlated with HPLC values and variations (usually over-estimates) were consistent with effects of light intensity and nutrient availability on fluorescence quenching. AOA estimates of taxonomic structure were consistent with those from HPLC-derived marker pigments by ChemTax, with both methods indicating domination by chromophytes and green algae in North Inlet and chromophytes and cyanobacteria in the Neuse.

Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system.

Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter.

Small phytoplankton and carbon export from the surface ocean.

Autotrophic picoplankton dominate primary production over large oceanic regions but are believed to contribute relatively little to carbon export from surface layers. Using analyses of data from the equatorial Pacific Ocean and Arabian Sea, we show that the relative direct and indirect contribution of picoplankton to export is proportional to their total net primary production, despite their small size. We suggest that all primary producers, not just the large cells, can contribute to export from the surface layer of the ocean at rates proportional to their production rates.