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方法之一 研究工具。这些技术利用大规模多重单分子 RNA 成像来提供 不仅可以直接测量完整样本中每个细胞的表达谱，还可以直接测量每个细胞的位置 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并发现和编目细胞类型、状态和 谱系具有高分辨率荧光显微镜来询问分子组织的能力 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 分子成像有望在众多主题中取得进展，从细胞内 RNA 组织在细胞内的作用开始 突触重塑，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，仅举几个例子。 一种基于图像的单细胞转录组学技术——MERFISH（多重误差稳健荧光 原位杂交）——由于其高分辨率、高捕获效率，已成为一项领先技术， 单分子灵敏度、无与伦比的通量以及经过验证的细胞内图谱能力 组织大部分转录组并发现、功能注释和绘制其中的细胞类型 完整的组织。然而，MERFISH 仍然是一项新兴技术，需要充分释放变革潜力 一般来说，对于 MERFISH 和空间分辨单细胞转录组学来说，这项技术必须成熟。 首先，MERFISH 必须进行全转录组分析。多重化不是障碍，而是几个RNA 类别——高表达 RNA、短 RNA 和高度同源 RNA——对此仍然具有挑战性 技术。通过新的实验和计算进步的结合，我们将扩展 MERFISH 针对这些类别，创建全转录组 MERFISH 并允许无假设的发现。 其次，对单细胞通量的生物学需求是惊人的，因为即使是小组织也常常含有 数千万个细胞。通过结合新的样品制备技术，一种新兴的超高通量方法 吞吐量显微镜，以及先进的图像存储和分析工具，我们将提高吞吐量 MERFISH 数量级，可对大组织区域和数千万个细胞进行表征。 最后，全转录组成像的变革潜力可能非常广泛，但 MERFISH 已经 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和 质量指标使 MERFISH 应用于所有组织和生物体成为常规。 在这里，我们将通过提供快速、稳健和常规的整体来释放这种新兴技术的潜力。 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 通用工具可以促进一系列真正卓越的基础和转化生物医学研究。

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.