Development and validation of an imaging cell sorter for integrated single cell genome and morphology analyses

海洋生态系统由微小的单细胞生物主导。由于在实验室条件下培养的普遍抗性和海洋微生物的惊人多样性，分子研究工具正在成为越来越重要的信息来源，了解它们的组成和功能，对全球环境变化和人为污染物的响应，以及它们作为生物能源、制药和生物技术应用的天然产物来源的潜力。然而，尽管分子工具具有强大的绝对吞吐量和不受培养偏差的影响，但它们所揭示的基因组序列与这些序列所代表的生物学特征（如细胞形态）之间的连通性往往较弱。该项目的目标是开发和验证下一代荧光激活细胞分选器，这将有助于弥合这一差距。该仪器将能够捕获同一单个细胞的图像和DNA。它还将介绍一般流式细胞术技术的几个额外改进。单细胞基因组学与成像的结合将使我们能够更深入地了解自然界中未培养的微生物群的组成和作用，并在当代分子数据集和基于细胞形态学的浮游生物研究的悠久历史之间提供关键的、缺失的联系。目前，市场上还没有合适的仪器来完成这项任务。该仪器将在实验室条件下和考察船上进行验证和优化。通过核心设施服务、讲习班、出版物和潜在的商业化，研究界将很容易获得这项新技术。流式细胞术和细胞分选技术在海洋浮游生物的定量、光学表征、靶向培养以及分子和元素分析等方面发挥着重要作用。该项目的目标是开发和验证下一代荧光激活细胞分选器，与现有仪器相比，该分选器包含以下主要技术改进：a)使用新型光学几何结构捕获分选细胞的高质量图像；B)细胞荧光作用光谱分析；C)光散射和荧光信号的信噪比大大提高。这些技术进步将使人们能够对主宰海洋生态系统的各种未培养的小型真核生物、细菌和古细菌的基因组、形态、大小和单个细胞、菌落和聚集体的基因组、形态、大小和色素进行高通量综合分析。新仪器还将有助于无分选细胞分析和分选门的描绘，而不需要费力和不精确的显微镜分选。该仪器的性能将在实验室条件和研究船上的各种操作模式下进行验证和优化。为了确保这项新技术能够被广泛的研究团体使用，它的使用将被纳入Bigelow海洋科学实验室的单细胞基因组学中心和J.J. MacIsaac水生细胞术设施的标准服务中。这些核心设施已经帮助支持了全球100多个组织的研究。还将探讨仪器商业化的各种选择。一名博士后研究员和一名本科生将从事仪器的开发和验证。为了促进新技术的进一步传播和讨论，该项目将为毕格罗实验室第四届微生物单细胞基因组学研讨会和年度水生流式细胞术课程提供部分支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Marine ecosystems are dominated by microscopic, unicellular life. Due to the prevalent resistance to cultivation under laboratory conditions and staggering diversity of marine microorganisms, molecular research tools are becoming increasingly important sources of information about their composition and function, response to global environmental changes and anthropogenic contaminants, as well as their potential as sources of natural products for bioenergy, pharmaceutical and biotech applications. However, although powerful in their sheer throughput and independence from cultivation biases, molecular tools often suffer from weak connectivity between the genomic sequences that they reveal and the biological features that these sequences represent, such as cell morphology. The goal of this project is to develop and validate a next-generation fluorescence-activated cell sorter that will help bridging this gap. The instrument will enable the capture of images and DNA of the same, individual cell. It will also introduce several additional improvements in the general flow cytometry technology. The integration of single cell genomics with imaging will enable deeper insights into the composition and roles of uncultured microbial groups in nature and provide the critical, missing link between the contemporary, molecular data sets and the long history of cell morphology-based plankton studies. Currently, there is no suitable instrument on the market for this task. The instrument will be validated and optimized under laboratory conditions and onboard a research vessel. The new technology will be made readily accessible to the research community through core facility services, workshops, publications and potential commercialization. Flow cytometry and cell sorting play important roles in the quantification, optical characterization, targeted cultivation, as well as molecular and elemental analyses of marine plankton. The goal of this project is to develop and validate a next-generation fluorescence-activated cell sorter that incorporates the following, major technical improvements as compared to the existing instruments: a) high-quality images of the sorted cells are captured using novel optical geometry; b) cell fluorescence action spectra are analyzed; c) signal-to-noise ratios are greatly improved for light scatter and fluorescence signals. These technological advances will enable high-throughput, integrated analyses of genomes, morphologies, sizes and pigments of individual cells, colonies and aggregates of the various uncultured lineages of small eukaryotes, bacteria and archaea that dominate marine ecosystems. The new instrument will also be instrumental in sort-free cell analyses and in delineation of the sort gate without the need for the laborious and imprecise bulk sorting for microscopy. The performance of this instrument will be validated and optimized under various operational modes in laboratory conditions and onboard a research vessel. To ensure easy access to this new technology to the broad research community, its use will be incorporated into standard services of the Single Cell Genomics Center and the J.J. MacIsaac Facility for Aquatic Cytometry at Bigelow Laboratory for Ocean Sciences. These core facilities already helped supporting research at over a hundred organizations around the globe. Options for instrument commercialization will also be explored. One post-doctoral researcher and one undergraduate student will be engaged in the development and validation of the instrument. To foster further dissemination and discussion of the new technology, this project will provide partial support for the fourth microbial single cell genomics workshop and annual aquatic flow cytometry courses at Bigelow Laboratory.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.