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

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领域之一， 研究工具。这些技术利用大规模多路复用单分子RNA成像， 不仅直接测量完整样品中每个细胞的表达谱，而且还测量每个细胞的位置， RNA分子。因此，这些技术联合收割机结合了单细胞RNA测序的能力 生成全转录组表达测量，发现和分类细胞类型，状态， 谱系的能力，高分辨率，荧光显微镜询问的分子组织， 细胞，定义它们的形态，并揭示它们的相互作用和组织。因此，原位转录组规模 分子成像有望在一系列广泛的主题中取得进展，从细胞内RNA组织的作用， 突触重构对宿主微生物群落空间结构的影响及其对宿主基因的影响 表达，到肿瘤发生中微环境的调节作用，仅举几个例子。 一种基于图像的单细胞转录组学技术-MERFISH（多路错误鲁棒荧光 原位杂交）-由于其高分辨率，高捕获效率， 单分子灵敏度和无与伦比的通量，结合其经证实的绘制细胞内 组织转录组的大部分，并发现，功能注释和映射细胞类型 完整的组织然而，MERFISH仍然是一项新兴技术，要充分释放变革潜力， 对于MERFISH和空间分辨单细胞转录组学而言，该技术必须成熟。 首先，MERFISH必须是全转录组。多重化不是障碍，而是几个RNA 高表达的RNA、短RNA和高度同源的RNA这几类RNA仍然是这方面的挑战 法通过结合新的实验和计算进展，我们将扩展MERFISH 这些类别，创建全转录组MERFISH并允许无假设发现。 其次，单细胞通量的生物学需求是惊人的，因为即使是小组织也经常含有 数以千万计的细胞。通过结合新的样品制备技术， 通过显微镜和先进的图像存储和分析工具，我们将提高 MERFISH的数量级，允许表征大的组织区域和数千万个细胞。 最后，全转录组成像的变革潜力可能非常广泛，但MERFISH具有 仅在少数组织中得到验证。因此，我们将提供一套强大的样品制备方案， 将MERFISH应用于所有组织和生物体的常规质量指标。 在这里，我们将释放这种新兴技术的潜力，提供快速，强大的，和常规的整体- 转录组MERFISH。由于基因表达是生命各个领域细胞身份和行为的关键，因此， 通用工具可以使一个真正了不起的基础和转化生物医学研究范围。

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.