A fluorescence-activated cell sorting subsystem for the Imaging FlowCytobot

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Limnology and Oceanography: Methods 15 (2017): 94–102, doi:10.1002/lom3.10145.Recent advances in plankton ecology have brought to light the importance of variability within populations and have suggested that cell-to-cell differences may influence ecosystem-level processes such as species succession and bloom dynamics. Flow cytometric cell sorting has been used to capture individual plankton cells from natural water samples to investigate variability at the single cell level, but the crude taxonomic resolution afforded by the fluorescence and light scattering measurements of conventional flow cytometers necessitates sorting and analyzing many cells that may not be of interest. Addition of imaging to flow cytometry improves classification capability considerably: Imaging FlowCytobot, which has been deployed at the Martha's Vineyard Coastal Observatory since 2006, allows classification of many kinds of nano- and microplankton to the genus or even species level. We present in this paper a modified bench-top Imaging FlowCytobot (IFCB-Sorter) with the capability to sort both single cells and colonies of phytoplankton and microzooplankton from seawater samples. The cells (or subsets selected based on their images) can then be cultured for further manipulation or processed for analyses such as nucleic acid sequencing. The sorting is carried out in two steps: a fluorescence signal triggers imaging and diversion of the sample flow into a commercially available “catcher tube,” and then a solenoid-based flow control system isolates each sorted cell along with 20 μL of fluid.NSF Grant Number: OCE-11300140; WHOI internal support; NSERC through a Post-Graduate Masters awar

Microzooplankton community structure investigated with imaging flow cytometry and automated live-cell staining

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Marine Ecology Progress Series 550 (2016): 65-81, doi:10.3354/meps11687.Protozoa play important roles in grazing and nutrient recycling, but quantifying these roles has been hindered by difficulties in collecting, culturing, and observing these often-delicate cells. During long-term deployments at the Martha’s Vineyard Coastal Observatory (Massachusetts, USA), Imaging FlowCytobot (IFCB) has been shown to be useful for studying live cells in situ without the need to culture or preserve. IFCB records images of cells with chlorophyll fluorescence above a trigger threshold, so to date taxonomically resolved analysis of protozoa has presumably been limited to mixotrophs and herbivores which have eaten recently. To overcome this limitation, we have coupled a broad-application ‘live cell’ fluorescent stain with a modified IFCB so that protozoa which do not contain chlorophyll (such as consumers of unpigmented bacteria and other heterotrophs) can also be recorded. Staining IFCB (IFCB-S) revealed higher abundances of grazers than the original IFCB, as well as some cell types not previously detected. Feeding habits of certain morphotypes could be inferred from their fluorescence properties: grazers with stain fluorescence but without chlorophyll cannot be mixotrophs, but could be either starving or feeding on heterotrophs. Comparisons between cell counts for IFCB-S and manual light microscopy of Lugol’s stained samples showed consistently similar or higher counts from IFCB-S. We show how automated classification through the extraction of image features and application of a machine-learning algorithm can be used to evaluate the large high-resolution data sets collected by IFCBs; the results reveal varying seasonal patterns in abundance among groups of protists.This research was supported in part by NSF (grants OCE-1130140, OCE-1434440), NASA (grants NNX11AF07G and NNX13AC98G), the Gordon and Betty Moore Foundation (grants 934 and 2649), and the Woods Hole Oceanographic Institution’s Innovative Technology Program

Diatoms favor their younger daughters

Author Posting. © Association for the Sciences of Limnology and Oceanography, 2012. This article is posted here by permission of Association for the Sciences of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 57 (2012): 1572-1578, doi:10.4319/lo.2012.57.5.1572.We used a time-lapse imaging approach to examine cell division in the marine centric diatom Ditylum brightwellii and observed that daughter cells who inherited their parents' hypothecal frustule half were more likely to divide before their sisters. This is consistent with observations in Escherichia coli of a bias between sister cells, where faster growth in one sister is thought to arise from its inheriting parental material with less oxidative damage. We also observed that hypothecal sisters in D. brightwellii were more likely to inherit a greater proportion of their parents' cellular material, similar to what has been seen in E. coli. We found a statistically significant correlation between the amount of parental material inherited by a hypothecal daughter and its relative division rate, indicating that this extra material inherited by the hypothecal daughter plays a role in its more rapid division. Furthermore, the intercept in this regression was greater than zero, indicating that other factors, such as differences in the quality of inherited material, also play a role. This similarity between two taxonomically distant microbes suggests that favoritism toward one daughter might occur broadly among unicellular organisms that reproduce asexually by binary fission. Such a bias in cell division might be advantageous, given model predictions that show that favoring one daughter at the expense of the other can result in higher population growth rates, increasing the chance that a cell's genotype will survive compared to a model where the daughters divide at equal rates.This research was funded in part by the Woods Hole Oceanographic Institution through an Ocean Life Institute Postdoctoral Scholarship to S.R.L. and by support to R.J.O. and H.M.S. from the Gordon and Betty Moore Foundation

Pump-During-Probe Fluorometry of Phytoplankton: Group-Specific Photosynthetic Characteristics from Individual Cell Analysis

Saturating-flash fluorescence techniques are used to monitor the state of the photosynthetic apparatus in phytoplankton under natural conditions. At present these are bulk water measurements, which produce estimates of average properties of all the fluorescent particles present in a sample. Here we describe an improved approach for single-cell measurements of phytoplankton. We have combined individual- cell 'pump-during-probe' measurements of chlorophyll fluorescence induction on the time scale of 30 to 100 microseconds with flow cytometric characterization of each cell, to obtain population-specific photosynthetic characteristics. The results provide information about the potential quantum yield of photochemistry, the fraction of functional reaction centers, and the functional absorption cross section for photosystem 2

Bio-optical discrimination of diatoms from other phytoplankton in the surface ocean: Evaluation and refinement of a model for the Northwest Atlantic

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Remote Sensing of Environment 217 (2018): 126-143, doi:10.1016/j.rse.2018.08.010.Diatoms dominate global silica production and export production in the ocean; they form the base of productive food webs and fisheries. Thus, a remote sensing algorithm to identify diatoms has great potential to describe ecological and biogeochemical trends and fluctuations in the surface ocean. Despite the importance of detecting diatoms from remote sensing and the demand for reliable methods of diatom identification, there has not been a systematic evaluation of algorithms that are being applied to this end. The efficacy of these models remains difficult to constrain in part due to limited datasets for validation. In this study, we test a bio-optical algorithm developed by Sathyendranath et al. (2004) to identify diatom dominance from the relationship between ratios of remote sensing reflectance and chlorophyll concentration. We evaluate and refine the original model with data collected at the Martha's Vineyard Coastal Observatory (MVCO), a near-shore location on the New England shelf. We then validated the refined model with data collected in Harpswell Sound, Maine, a site with greater optical complexity than MVCO. At both sites, despite relatively large changes in diatom fraction (0.8–82% of chlorophyll concentration), the magnitude of variability in optical properties due to the dominance or non-dominance of diatoms is less than the variability induced by other absorbing and scattering constituents of the water. While the original model performance was improved through successive re-parameterizations and re-formulations of the absorption and backscattering coefficients, we show that even a model originally parameterized for the Northwest Atlantic and re-parameterized for sites such as MVCO and Harpswell Sound performs poorly in discriminating diatom-dominance from optical properties.This work was supported by: a Woods Hole Oceanographic Institution Summer Student Fellowship (NSF REU award #1156952) and a Bowdoin College Grua/O'Connell Research Award to SJK; grants to HMS from NASA (Ocean Biology and Biogeochemistry program and Biodiversity and Ecological Forecasting program), NSF (Ocean Sciences), the Gordon and Betty Moore Foundation, the Simons Foundation, and NOAA through the Cooperative Institute for the North Atlantic Region (CINAR) under Cooperative Agreement NA14OAR4320158; and grants to CSR from NASA (Ocean Biology and Biogeochemistry program)

Streaming Gaussian Dirichlet Random Fields for Spatial Predictions of High Dimensional Categorical Observations

We present the Streaming Gaussian Dirichlet Random Field (S-GDRF) model, a novel approach for modeling a stream of spatiotemporally distributed, sparse, high-dimensional categorical observations. The proposed approach efficiently learns global and local patterns in spatiotemporal data, allowing for fast inference and querying with a bounded time complexity. Using a high-resolution data series of plankton images classified with a neural network, we demonstrate the ability of the approach to make more accurate predictions compared to a Variational Gaussian Process (VGP), and to learn a predictive distribution of observations from streaming categorical data. S-GDRFs open the door to enabling efficient informative path planning over high-dimensional categorical observations, which until now has not been feasible.Comment: 10 pages, 5 figures. Published in Springer Proceedings of Advanced Robotics, ISER 2023 Conference Proceeding

Imaging FlowCytobot modified for high throughput by in-line acoustic focusing of sample particles

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Limnology and Oceanography: Methods 15 (2017): 867–874, doi:10.1002/lom3.10205.Imaging FlowCytobot, a submersible instrument that measures optical properties and captures images of nano- and microplankton-sized particles, has proved useful in plankton studies, but its sampling rate is limited by the ability of hydrodynamic focusing to accurately position flowing sample particles. We show that IFCB's sampling rate can be increased at least several-fold by implementing in-line acoustic focusing upstream of the flow cell. Particles are forced to the center of flow by acoustic standing waves created by a piezo-electric transducer bonded to the sample capillary and driven at the appropriate frequency. With the particles of interest confined to the center of the sample flow, the increased size of the sample core that accompanies increased sample flow rate no longer degrades image and signal quality as it otherwise would. Temperature affects the optimum frequency (through its effect on the speed of sound in water), so a relationship between sample temperature and optimum frequency for acoustic focusing was determined and utilized to control the transducer. The modified instrument's performance was evaluated through analyses of artificial particles, phytoplankton cultures, and natural seawater samples and through deployments in coastal waters. The results show that large cells, especially dinoflagellates, are acoustically focused extremely effectively (which could enable, for example, > 10-fold increased sampling rate of harmful algal bloom species, if smaller cells are ignored), while for nearly all cell types typically monitored by IFCB, threefold faster data accumulation was achieved without any compromises. Further increases are possible with more sophisticated software and/or a faster camera.NSF Grant Numbers: OCE-1130140 , OCE-113113

Leader honesty/humility and subordinate organizational citizenship behavior: a case of too-much-of-a-good-thing?

Purpose – On the basis of theories of social cognition and moral identity and the meta-theoretical principle of “too-much-of-a-good-thing,” the purpose of this study is to develop and test a model that explains when and why leader honesty/humility promotes subordinate organizational citizenship behavior directed at individuals (OCBI) as mediated through subordinate moral identity centrality. Design/methodology/approach – In this field study, with online surveys, multisource data were collected from 218 United States Air Force officers and their subordinates. Data were analyzed with MEDCURVE SPSS macro tools. Findings – A nonlinear indirect effect of leader honesty/humility on subordinate OCBI through subordinate moral identity centrality was found. This conditional indirect effect occurred through a curvilinear (inverted Ushape) relationship between leader honesty/humility and subordinate moral identity centrality and a positive linear relationship between subordinate moral identity centrality and OCBI. Research limitations/implications – Cross-sectional data were collected. Future research might replicate findings using experimental and longitudinal designs. Practical implications – Recruiting and selecting leaders who possess a moderate level of honesty/humility may serve as the first step in producing prosocial behavior during social interactions with subordinates. Originality/value – This study extends the literature on character and leadership by applying the too-muchof-a-good-thing principle to empirically test the complex nature of the relationship between

Mesoscale variability in intact and ghost colonies of Phaeocystis antarctica in the Ross Sea : distribution and abundance

© The Author(s), 2016. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Marine Systems 166 (2017): 97-107, doi:10.1016/j.jmarsys.2016.05.007.Phaeocystis, a genus with a cosmopolitan distribution and a polymorphic life cycle, was observed during summer in the Ross Sea, Antarctica, where large blooms of this haptophyte regularly occur. The mesoscale vertical and horizontal distributions of colonies of P. antarctica were assessed using a towed Video Plankton Recorder (VPR). The mean size of colonies was 1.20 mm, and mean abundances within the three VPR surveys were 4.86, 1.96, and 11.5 mL-1. In addition to the typical spherical, transparent colonies, the VPR quantified an optically dissimilar form of colony that had a distinctive translucent appearance. It also measured the abundance of collapsed colonies, similar to those observed previously from cultures and mesocosms, which we called “ghost colonies”. The translucent colonial form had a different distribution than the more common colonial form, and at times was more abundant. Relative to intact colonies, the ghost colonies occurred less frequently, with mean abundances in the three surveys being 0.01, 0.08, and 0.0004 mL-1. Ghost colonies generally were found below the euphotic zone, where they often were in greater abundance than intact colonies. However, the relationship of ghost colonies to intact P. antarctica colonies was not direct or consistent, suggesting that the formation of ghost colonies from living colonies and their appearance within the water column were not tightly coupled. Given their relative scarcity and low carbon content, it is unlikely that ghost colonies contribute substantially to vertical flux; however, it is possible that we did not sample periods of major flux events, and as a result minimized the importance of ghost colonies to vertical flux. They do, however, represent a poorly documented feature of polar haptophyte life cycles.This research was supported by grants from the National Science Foundation (ANT-0944254 and ANT-0944165). HMS and EEP acknowledge support of the Gordon and Betty Moore Foundation (Grant #2649) for image informatics development.2018-06-0

Envisioning a marine biodiversity observation network

Author Posting. © University of California Press and American Institute of Biological Sciences, 2013. This article is posted here by permission of University of California Press and American Institute of Biological Sciences for personal use, not for redistribution. The definitive version was published in BioScience 63 (2013): 350-361, doi:10.1525/bio.2013.63.5.8.Humans depend on diverse ocean ecosystems for food, jobs, and sustained well-being, yet many stressors threaten marine life. Extensive research has demonstrated that maintaining biodiversity promotes ocean health and service provision; therefore, monitoring the status and trends of marine biodiversity is important for effective ecosystem management. However, there is no systematic sustained program for evaluating ocean biodiversity. Coordinating existing monitoring and building a proactive marine biodiversity observation network will support efficient, economical resource management and conservation and should be a high priority. A synthesis of expert opinions suggests that, to be most effective, a marine biodiversity observation network should integrate biological levels, from genes to habitats; link biodiversity observations to abiotic environmental variables; site projects to incorporate environmental forcing and biogeography; and monitor adaptively to address emerging issues. We summarize examples illustrating how to leverage existing data and infrastructure to meet these goals