Rapid, Robust, and Routine: Multiplexed Microscopy for Spatially Resolved Whole-Transcriptomic Single-Cell Profiling and the Construction of Cell Atlases of all Tissues and in all Organisms

快速、稳健和常规：用于空间分辨全转录组单细胞分析和所有组织和所有生物体细胞图谱构建的多重显微镜

• 批准号: 10278148
• 负责人: Jeffrey Moffitt
• 金额: $ 42.92万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10278148
• 关键词: Address; Adoption; Algorithms; Area; Atlases; Bar Codes; Behavior; Biological; Biomedical Research; Catalogs; Categories; Cell Count; Cell physiology; Cells; Clinical; Color; DNA; Data Analyses; Data Collection; Data Compression; Excision; Exons; Fluorescence; Fluorescence Microscopy; Fluorescent in Situ Hybridization; Formalin; Gene Expression; Genome; Image; Image Compression; In Situ; Individual; Kinetics; Life; Location; Maps; Measurement; Measures; Methodology; Methods; Microscopy; Modification; Molecular; Morphology; Names; Organism; Paraffin Embedding; Performance; Phylogenetic Analysis; Preparation; Protein Isoforms; Protocols documentation; Quality Control; RNA; RNA Splicing; Resolution; Retrieval; Role; Sampling; Scheme; Signal Transduction; Speed; Stains; Synapses; Techniques; Technology; Time; Tissue Embedding; Tissue Harvesting; Tissues; Variant; base; cell behavior; cell type; cost; design; imaging approach; improved; microbial community; microscopic imaging; molecular imaging; molecular scale; novel; novel strategies; sample archive; sample fixation; single molecule; single-cell RNA sequencing; submicron; tissue preparation; tool; transcriptome; transcriptomics; tumorigenesis

Image-based approaches to single-cell transcriptomics represent one of the most exciting emerging biomedical research tools. These technologies leverage massively multiplexed single-molecule RNA imaging to provide a direct measure of not just the expression profile of every cell within intact samples but also the location of every RNA molecule within those cells. As such, these techniques combine the ability of single-cell RNA sequencing to generate whole-transcriptome expression measurements and discover and catalog cell types, states, and lineage with the ability of high-resolution, fluorescence microscopy to interrogate the molecular organization of cells, define their morphology, and reveal their interactions and organization. Thus, in situ transcriptome-scale molecular imaging promises advances in a vast array of topics, from the role of intracellular RNA organization in synaptic remodeling, to the spatial organization of commensal microbial communities and its effect on host gene expression, to the modulatory role of the microenvironment in tumorigenesis, to name only a few examples. One image-based single-cell transcriptomics technique—MERFISH (multiplexed error robust fluorescence in situ hybridization)—has emerged as a leading technology given its high resolution, high capture efficiency, single-molecule sensitivity, and unparalleled throughput combined with its proven ability to map the intracellular organization of large fractions of the transcriptome and discover, functionally annotate, and map cell types within intact tissues. However, MERFISH remains a nascent technology, and to fully unlock the transformative potential of both MERFISH and spatially resolved single-cell transcriptomics in general, this technology must be matured. First, MERFISH must be made whole-transcriptome. Multiplexing is not the barrier, rather several RNA categories—highly expressed RNAs, short RNAs, and highly homologous RNAs—remain challenging for this technique. Through a combination of new experimental and computational advances, we will extend MERFISH to these categories, creating whole-transcriptome MERFISH and allowing hypothesis-free discovery. Second, the biological demands for single-cell throughput are staggering, as even small tissues often contain tens of millions of cells. By combining new sample preparation techniques, an emerging approach to ultra-high- throughput microscopy, and advanced image storage and analysis tools, we will increase the throughput of MERFISH by orders of magnitude, allowing characterization of large tissue areas and tens of millions of cells. Finally, the transformative potential for whole-transcriptome imaging could be very broad, yet MERFISH has been validated in only a few tissues. Thus, we will provide a robust suite of sample preparation protocols and quality metrics to make routine the application of MERFISH to all tissues and organisms. Here we will unlock the potential of this emerging technique by delivering rapid, robust, and routine whole- transcriptome MERFISH. As gene expression is key to cellular identity and behavior in all domains of life, this general tool could empower a truly remarkable range of basic and translational biomedical research.

基于图像的单细胞转录组学的方法代表了最令人兴奋的新兴生物医学之一 研究工具。这些技术利用大量多重的单分子RNA成像提供 直接测量完整样本中每个单元的表达式曲线，还可以直接测量每个单元格的位置 这些细胞内的RNA分子。因此，这些技术结合了单细胞RNA测序的能力 生成全转录组表达测量结果，并发现和目录细胞类型，状态和 谱系具有高分辨率，荧光显微镜质疑分子组织的能力 细胞，定义其形态，并揭示其相互作用和组织。那，原位转录组尺度 分子成像有望从细胞内RNA组织在 突触重塑，向共生微生物群落的空间组织及其对宿主基因的影响 表达微环境在肿瘤发生中的调节作用，仅列举了几个例子。 一种基于图像的单细胞转录组学技术 - cerfish（多重误差可靠荧光 原位杂交） - 鉴于其高分辨率，高捕获效率，已成为领先的技术， 单分子灵敏度和无与伦比的吞吐量与其验证的细胞内绘制能力相结合 组织转录组的大分子并发现，功能上注释和映射单元格中的类型 完整的组织。但是，Merfish仍然是一种新生的技术，并充分释放了变革性的潜力 通常，在Merfish和空间分辨的单细胞转录组学中，该技术必须成熟。 首先，必须将Merfish制作全转录组。多路复用不是障碍，而是几个RNA 类别 - 高度表达的RNA，简短的RNA和高度同源的RNA-对此挑战 技术。通过新的实验和计算进步的结合，我们将扩展Merfish 对于这些类别，创建全转录组MERFISH并允许无假设的发现。 其次，对单细胞吞吐量的生物学需求令人震惊，因为即使是小组织也经常包含 数千万细胞。通过结合新样品制备技术，这是一种超高的新兴方法 吞吐量显微镜以及高级图像存储和分析工具，我们将增加 Merfish通过数量级，允许表征大组织区域和数千万细胞。 最后，全转录组成像的变革潜力可能非常广泛，但Merfish具有 仅在几个组织中得到验证。这是，我们将提供一套可靠的样本准备协议的套件，并提供 质量指标使常规的Merfish应用于所有组织和生物。 在这里，我们将通过提供快速，健壮和常规的整体 - 转录组Merfish。由于基因表达是生命所有领域中细胞身份和行为的关键，因此 一般工具可以增强一系列基本和翻译的生物医学研究的能力。