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

用于集成单细胞基因组和形态分析的成像细胞分选仪的开发和验证

• 批准号: 1829879
• 负责人: Ramunas Stepanauskas
• 金额: $ 139.63万
• 依托单位: Bigelow Laboratory for Ocean Sciences
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1829879&HistoricalAwards=false
• 关键词: Development; validation; imaging; cell; sorter

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.

海洋生态系统由微观的单细胞生命主导。由于对实验室条件下培养的普遍抵制和海洋微生物惊人的多样性，分子研究工具正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反应，以及它们作为生物能源、制药和生物技术应用天然产品来源的潜力。然而，尽管分子工具在其绝对通量和独立于培养偏差方面很强大，但它们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）之间的连接性往往很弱。该项目的目标是开发和验证下一代荧光激活细胞分选仪，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的改进。单细胞基因组学与成像的整合将使人们能够更深入地了解自然界中未培养微生物群体的组成和作用，并提供当代分子数据集与基于细胞形态学的浮游生物研究的悠久历史之间关键的缺失环节。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研究船上进行验证和优化。新技术将通过核心设施服务、讲习班、出版物和潜在的商业化，使研究界能够随时获得。流式细胞术和细胞分选在海洋浮游生物的定量、光学特性、定向培养以及分子和元素分析中发挥着重要作用。该项目的目标是开发和验证下一代荧光激活细胞分选仪，与现有仪器相比，该分选仪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）分析细胞荧光作用光谱; c）大大提高光散射和荧光信号的信噪比。这些技术进步将能够对海洋生态系统中占主导地位的各种未培养的小型真核生物、细菌和古菌谱系的单个细胞、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划分，而无需进行费力且不精确的显微镜批量分选。该仪器的性能将在实验室条件下和研究船上的各种操作模式下进行验证和优化。为了确保广泛的研究界容易获得这项新技术，它的使用将被纳入毕格罗海洋科学实验室的单细胞基因组学中心和J. J. MacIsaac水生细胞计数设施的标准服务。这些核心设施已经帮助支持了地球仪100多个组织的研究。还将探讨仪器商业化的备选方案。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，该项目将为第四届微生物单细胞基因组学研讨会和毕格罗实验室的年度水生流式细胞术课程提供部分支持。该奖项反映了NSF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。