The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy. Using antibodies labeled with short DNA oligonucleotides, multiple targets are visualized successively by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. The structural integrity of the tissue is maintained owing to only a single labelling step during sample preparation. Multiple targets are imaged using a single laser wavelength, minimizing chromatic aberration. This method proved robust for multi-target imaging in semi-thin tissue sections, paving the way towards structural cell biology with single-molecule super-resolution microscopy.

中文翻译：

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DNA辅助单分子定位显微镜观察神经元突触处的多蛋白模式

超分辨率显微镜（SRM）的发展拓宽了我们对生物材料中生物分子结构和功能的理解。对单个区域内的多个目标进行成像将阐明它们相对于细胞基质和邻近生物分子的空间定位，从而揭示多蛋白大分子结构及其功能的相互依赖性。但是，SRM方法限于在单次采集过程中可以成像的合适的荧光团的数量，以及在抗体清洗和保留以进行有机染料多路复用时抗原的损失。我们报告使用DNA辅助单分子定位显微镜在350-400 nm厚的神经元组织切片中的突触前和突触内多个蛋白质目标的可视化。使用标记有短DNA寡核苷酸的抗体，通过顺序交换成像缓冲液中存在的荧光团标记的互补寡核苷酸，可以连续显示多个目标。由于样品制备过程中只有一个标记步骤，因此可以保持组织的结构完整性。使用单个激光波长对多个目标成像，以最小化色差。这种方法被证明对半薄组织切片中的多目标成像具有鲁棒性，这为单分子超分辨率显微镜为结构细胞生物学铺平了道路。使用单个激光波长对多个目标成像，以最小化色差。这种方法被证明对半薄组织切片中的多目标成像具有鲁棒性，这为单分子超分辨率显微镜为结构细胞生物学铺平了道路。使用单个激光波长对多个目标成像，以最小化色差。这种方法被证明对半薄组织切片中的多目标成像具有鲁棒性，这为单分子超分辨率显微镜为结构细胞生物学铺平了道路。