神经胶质瘤是组织学和遗传异质性肿瘤。然而经典的组织病理学分型往往忽视了肿瘤的高度异质性，无法满足精准病理诊断的要求。在此，提出了邻近锚定原位光谱编码放大（ProxISCA）用于RNA突变的多重成像，从而能够在单细胞和组织水平上对具有不同病理级别的脑胶质瘤进行视觉分型。基于连接的锁式探针可以区分单核苷酸变异，邻近引物的设计使扩增子能够锚定在目标RNA上，从而提高定位精度。基于DNA模块的光谱编码策略可以通过一次性标记显着提高RNA突变成像的多重能力，且成本低、操作简单。一靶一扩增子扩增使 ProxISCA 能够以单分子分辨率量化 RNA 突变拷贝数。基于这种方法，发现恶性程度较高的胶质瘤在细胞和组织水平上表达更多具有高相关性的基因，并表现出更大的细胞异质性。 ProxISCA为胶质瘤研究和精准诊断提供了工具，可以揭示细胞异质性与胶质瘤发生或发展之间的关系，并协助病理预后。© 2023 作者。河南大学和约翰·威利出版的《探索》

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.© 2023 The Authors. Exploration published by Henan University and John Wiley & Sons Australia, Ltd.