In situ digital holographic microscopy for rapid detection and monitoring of the harmful dinoflagellate, Karenia brevis.

Karenia brevis blooms, also known as red tide, are a recurring problem in the coastal Gulf of Mexico. These blooms have the capacity to inflict substantial damage to human and animal health as well as local economies. Thus, monitoring and detection of K. brevis blooms at all life stages and cell concentrations is essential for ensuring public safety. Current K. brevis monitoring methods have several limitations, including size resolution limits and concentration ranges, limited capacity for spatial and temporal profiling, and/or small sample volume processing. Here, a novel monitoring method wherein an autonomous digital holographic imaging microscope (AUTOHOLO), that overcomes these limitations and can characterize K. brevis concentrations in situ, is presented. Using the AUTOHOLO, in situ field measurements were conducted in the coastal Gulf of Mexico during an active K. brevis bloom over the 2020-21 winter season. Surface and sub-surface water samples collected during these field studies were also analyzed in the lab using benchtop holographic imaging and flow cytometry for validation. A convolutional neural network was trained for automated classification of K. brevis at all concentration ranges. The network was validated with manual counts and flow cytometry, yielding a 90% accuracy across diverse datasets with varying K. brevis concentrations. The usefulness of pairing the AUTOHOLO with a towing system was also demonstrated for characterizing particle abundance over large spatial distances, which could potentially facilitate characterization of spatial distributions of K. brevis during bloom events. Future applications of the AUTOHOLO can include integration into existing HAB monitoring networks to enhance detection capabilities for K. brevis in aquatic environments around the world.

Optical backscattering and linear polarization properties of the colony forming cyanobacterium Microcystis.

Light scattering characteristics of the cyanobacterium Microcystis are investigated with numerical models for sphere aggregates. During summer bloom seasons, Microcystis is prevalent in many inland waters across the globe. Monitoring concentrations with remote sensing techniques requires knowledge of the inherent optical properties (IOPs), especially the backscattering properties of Microcystis cells and colonies in natural settings. In situ measurements in waters dominated by Microcystis blooms have previously detected extremely high backscattering ratios, i.e., bb/b>0.043 at 443 nm [1], the highest to our knowledge in the natural environment. These highbb/bvalues could hold promise as a diagnostic tool in identifying and monitoring Microcystis using optical approaches. However, it has been unclear how this type of optically 'soft' organic particle can generate such highbb/bvalues. In this study, the Multiple Sphere T-matrix (MSTM) model is used to calculate the IOPs of model colonies, including bb/b. Colony sizes in the model ranged from several cells to several hundred and both colony packing density and cell gas vacuole content were varied. Results are compared with model results for equivalent-volume spheres (EVS) and direct in situ measurements. Colony formation was required in the modeling to reproduce the high bb/bconsistent with in situ measurements. The combination of moderate to very dense colony (packing density >30%) and high gas vacuole content in individual cells (volume percentage >20%) was the most favorable condition leading to rapid increases in bb/bwith increasing number of cells Ncell of the colony. Significant linear correlations were observed betweenbb/b and Ncell1/3 for these colonies, wherebb/b increased beyond 0.04 once cell number reached about 1000 cells in the case with the most densely packed cells and highest gas vacuole content. Within commonly observed colony sizes (Ncell <106), colonies with high gas vacuole content exhibited bb/bvalues up to 0.055, consistent with direct measurements from Lake Erie. Polarized scattering was also of interest as a diagnostic tool, particularly with future Earth-orbiting polarimeters being deployed for the NASA Plankton, Aerosols, Cloud, ocean Ecosystem (PACE) mission. The Degree of Linear Polarization (DoLP), expressed by the ratio of two Mueller matrix elements-P12/P11, decreased with increasing colony cell number for Microcystis. Another ratio of two Mueller matrix elementsP22/P11, an index for nonsphericity, also decreased with increasing colony size. In addition to higher relative backscattering, greater colony packing density and larger gas vacuole sizes both led to lower DoLP peak magnitude and lowerP22/P11. An optical opposition feature due to constructive phase interference that was observed previously for cosmic dusts is also present for these modeled colonies, manifested by a narrow intensity peak and negative polarization dip near exact backscattering direction, gradually forming as colony size increases.

Evidence for ubiquitous preferential particle orientation in representative oceanic shear flows.

In situ measurements were undertaken to characterize particle fields in undisturbed oceanic environments. Simultaneous, co-located depth profiles of particle fields and flow characteristics were recorded using a submersible holographic imaging system and an acoustic Doppler velocimeter, under different flow conditions and varying particle concentration loads, typical of those found in coastal oceans and lakes. Nearly one million particles with major axis lengths ranging from ∼14 µm to 11.6 mm, representing diverse shapes, sizes, and aspect ratios were characterized as part of this study. The particle field consisted of marine snow, detrital matter, and phytoplankton, including colonial diatoms, which sometimes formed "thin layers" of high particle abundance. Clear evidence of preferential alignment of particles was seen at all sampling stations, where the orientation probability density function (PDF) peaked at near horizontal angles and coincided with regions of low velocity shear and weak turbulent dissipation rates. Furthermore, PDF values increased with increasing particle aspect ratios, in excellent agreement with models of spheroidal particle motion in simple shear flows. To the best of our knowledge, although preferential particle orientation in the ocean has been reported in two prior cases, our findings represent the first comprehensive field study examining this phenomenon. Evidence of nonrandom particle alignment in aquatic systems has significant consequences to aquatic optics theory and remote sensing, where perfectly random particle orientation and thus isotropic symmetry in optical parameters is assumed. Ecologically, chain-forming phytoplankton may have evolved to form large aspect ratio chains as a strategy to optimize light harvesting.