Next generation submersible flow cytometry for plankton studies: Extended dynamic range and orthogonal imaging

微生物占海洋初级生产力和生物量的大部分，它们的群落结构在很大程度上决定了更高营养水平的结构。对调节群落结构的因素的基本理解需要对微生物进行详细和持续的观察。该项目将通过加强对海洋浮游生物的自动化现场监测仪器来填补一个重要的空白。这项研究将贡献新的观测技术，不仅在海洋研究界，而且对地方、州和联邦资源管理人员都可能产生广泛的影响。鉴于持续的环境变化和有关人类对沿海地区影响增加的预测，在基于海洋生态系统的管理和减轻人类健康风险（例如与有害藻华有关的风险）方面存在许多新的挑战。以科学理解和观察为依据的战略至关重要，人们越来越认识到详细的生物数据集的价值。这类数据集将由该项目提供，将为社会提供维持人类赖以获取食物、水、能源和气候调节的自然生态系统所需的信息。该项目包括多层次教育和外联工作。本科生的研究经历将有助于促进海洋科学的多样性。来自高多样性社区和低收入家庭比例的K-12教师将参与旨在促进初中和高中课程中科学素养的专业发展活动。研究人员将生产下一代自动潜水流式细胞仪，以扩大观察微观浮游生物的能力。这项工作将直接建立在他们之前开发两种原位流式细胞仪的经验之上：一种是测量单个细胞的光散射和荧光特性（针对沿海水域常见的微小蓝藻和最小的真核浮游植物细胞），另一种是捕获较大细胞的图像，允许自动图像分析以确定分类关系。两个目标涉及到提高成像仪器的设计：(1)结合现有两种仪器的功能，使一台仪器可以研究几乎整个浮游植物群落；(2)通过捕获较大细胞的两个图像（正面和侧面视图）来增加基于图像的自动表征的能力。这两种方法都将利用声波聚焦技术（一种将细胞置于流动中的技术）来更快地处理水样。第三个目标是通过在伍兹霍尔港的实地测试来评估新仪器的能力，然后通过在玛莎葡萄园岛海岸天文台的机会巡航和部署来获得美国东北大陆架的高分辨率时空观测。

Microbes account for most of the primary productivity and biomass in the ocean, and their community structure determines in large part that of higher trophic levels. A fundamental understanding of the factors that regulate community structure requires detailed and sustained observations of the microscopic organisms. This project will fill an important gap by enhancing instrumentation for automated in situ monitoring of marine plankton. The research will contribute new observational technologies that are likely to have broad impact, not only within the ocean research community, but also for local, state, and federal resource managers. In light of on-going environmental change and related projections of increased human impact on coastal zones, there are many new challenges for marine ecosystem-based management and mitigation of human health risks, such as those associated with harmful algal blooms. Strategies informed by scientific understanding and observations are critical and there is growing recognition of the value of detailed biological data sets. Such data sets, as will be enabled by this project, will provide society the information needed to sustain natural ecosystems that humans rely upon for food, water, energy, and climate regulation. The project includes multilevel education and outreach efforts. Research experiences for undergraduates will help promote diversity in ocean science. K-12 teachers, drawn from communities with high diversity and proportion of low income households, will engage in professional development activities designed to promote science literacy in middle and high school curricula.The researchers will produce the next generation of automated submersible flow cytometers with expanded capabilities for observing microscopic plankton. The work will build directly on their previous experience developing two in situ flow cytometric instruments: one that measures individual-cell light scattering and fluorescence properties (targeting the tiny cyanobacteria common in coastal waters and the smallest eukaryotic phytoplankton cells), and a second instrument that captures images of larger cells, which allows automated image analysis to determine taxonomic affiliation. Two objectives involve enhancing the imaging instrument design: (1) combining the functions of the two current instruments, so that a single instrument can study nearly the entire phytoplankton community; and (2) increasing the power of image-based automated characterization by capturing two images (front and side views) of the larger cells. Both approaches will take advantage of acoustic focusing, a technique by which cells are positioned in flow, to process water samples faster than would otherwise be possible. A third objective is to evaluate the new instruments' capabilities through field tests in Woods Hole Harbor and then to acquire high-resolution spatial-temporal observations on the Northeast US Shelf through cruises-of-opportunity and deployments at the Martha's Vineyard Coastal Observatory.