Pérez-Garza Janeth, Orea Jairo, Deane Zachary, Raimondi Gianna, Tripp Rebecca, Charles Imani, Ostroff Linnaea
Department of Physiology and Neurobiology, University of Connecticut, Storrs, Conn.
Institute for the Brain and Cognitive Sciences, University of Connecticut, Storrs, Conn.
bioRxiv. 2024 Aug 19:2024.08.17.605585. doi: 10.1101/2024.08.17.605585.
The molecular organization of cells and tissue is challenging to study due to the inefficiency of multiplexed molecular labeling methods and the limited options for combining microscopy modalities in a single specimen, especially when high spatial resolution is needed. Here we describe ultraplex microscopy, which combines serial multiplexing, ultrathin sectioning, and reversible embedding to circumvent incompatibilities between labeling and imaging techniques, enhance resolution, and expand multiplexing capacity within and across modalities. Samples can be labeled with antibodies, RNA probes, and tissue stains for imaging by brightfield, epifluorescence, super-resolution, and electron microscopy without specialized reagents or materials. We demonstrate applications in brain tissue including molecular profiling of single cells and axonal boutons, high-resolution molecular colocalization, and correlative imaging of fluorescent proteins with confocal and ultraplex microscopy. The power and versatility of ultraplex microscopy will be valuable in addressing currently intractable experimental questions in many systems and contexts.
由于多重分子标记方法效率低下，且在单个样本中组合显微镜检查方式的选择有限，尤其是在需要高空间分辨率时，细胞和组织的分子组织研究颇具挑战性。在此，我们描述了超多重显微镜技术，它结合了连续多重标记、超薄切片和可逆包埋技术，以规避标记和成像技术之间的不兼容性，提高分辨率，并扩展不同模态内和跨模态的多重标记能力。无需特殊试剂或材料，样本就可以用抗体、RNA探针和组织染色剂进行标记，以通过明场、落射荧光、超分辨率和电子显微镜进行成像。我们展示了其在脑组织中的应用，包括单细胞和轴突终扣的分子谱分析、高分辨率分子共定位，以及利用共聚焦显微镜和超多重显微镜对荧光蛋白进行相关成像。超多重显微镜技术的强大功能和通用性对于解决当前许多系统和环境中难以处理的实验问题将具有重要价值。
