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: 10494105
• 负责人: Jeffrey Moffitt
• 金额: $ 34.79万
• 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/10494105
• 关键词: Address; Adoption; Algorithms; Area; Atlases; Bar Codes; Behavior; Biological; Biomedical Research; Brain; Catalogs; Categories; Cell Count; Cell Culture Techniques; 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方法代表了最令人兴奋的新兴生物医学之一