High-Throughput, Highly Multiplexed In Situ Proteomic Imaging of Human Tissues

摘要 对于高内容的映射来说，高复用能力是必不可少的。然而，光谱重叠和缺乏 正交标记法给传统的荧光成像带来了严重的限制。对于蛋白质成像，这些 通过非传统探头的多路传输检测和专门的 仪器，迭代序列抗体标记和成像，或通过DNA条形码的序列检测。 虽然所有这些技术在理论上都可以实现高复用度，但它们的成本有限 吞吐量。利用原位信号放大将大大减少曝光时间--从而减少成像时间 帧，允许高吞吐量以及更高的灵敏度。虽然存在放大方法，但它们有 没有被强健地多路复用超过5-8个空间重叠的目标。尚未完全满足的关键技术需求 实现HuBMAP视觉是一种高度复用的信号放大技术，可以同时放大 将数十个不同的目标扩展到数十到数百倍，从而实现高度多路复用、高吞吐量、就地 人体组织中蛋白质的成像。我们提出了这样一种原位信号放大方法，该方法基于一种新的 我们最近发表的分子机制。在新方法中，用多个探针(DNA条形码)进行染色 一次抗体)将同时进行，然后所有条形码将被同时延伸到原位的长串联。最后，将通过快速交换和成像循环顺序执行串联序列的映射，将吞吐量提高10倍，同时能够检测组织中的稀有目标。除蛋白质外，该方法还将适用于RNA和DNA(染色体)靶标。我们将把该方法与 商业上可用的自动染色和成像系统，并将其应用于通过 在HuBMAP社区内外广泛协作。

Summary High multiplexing capability is indispensable for high-content mapping. However, spectral overlap and lack of orthogonal labeling create severe limitations for conventional fluorescence imaging. For protein imaging, these limitations have been circumvented through multiplexed detection via unconventional probes and specialized instruments, iterative sequential antibody labeling and imaging, or sequential detection through DNA-barcoding. Although all of these techniques can theoretically achieve high multiplexing, they come at a cost of limited throughput. Utilizing in situ signal amplification would substantially reduce exposure - and thus imaging - time per frame, allowing for high throughput as well as improved sensitivity. Although amplification methods exist, they have not been robustly multiplexed beyond 5-8 spatially overlapping targets. A critical unmet technical need for fully realizing the HuBMAP vision is a highly multiplexed signal amplification technique that can simultaneously amplify tens of distinct targets by tens- to hundreds-fold, thereby enabling highly multiplexed, high throughput, in situ imaging of proteins in human tissues. We propose such an in situ signal amplification method, based on a novel molecular mechanism that we recently published. In the new method, staining with multiple probes (DNA-barcoded primary antibodies) will be performed simultaneously, and then all barcodes will be simultaneously extended into long concatemers in situ. Finally, mapping of concatemers will be sequentially performed through rapid exchange and imaging cycles, improving throughput by 10-fold while enabling detection of rare targets in tissues. Beyond proteins, the method will be applicable to RNA and DNA (chromosome) targets. We will integrate the method with commercially available, automated staining and imaging systems, and apply it to image diverse human tissues via broad collaboration within and beyond HuBMAP community.