Narayanasamy Kaarjel K, Stojic Aleksandar, Li Yunqing, Sass Steffen, Hesse Marina R, Deussner-Helfmann Nina S, Dietz Marina S, Kuner Thomas, Klevanski Maja, Heilemann Mike
Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany.
Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Frankfurt, Germany.
Front Synaptic Neurosci. 2021 Jun 17;13:671288. doi: 10.3389/fnsyn.2021.671288. eCollection 2021.
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 (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.
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。在单个区域内对多个靶点进行成像，将阐明它们相对于细胞基质和相邻生物分子的空间定位，揭示多蛋白大分子结构及其功能上的相互依赖关系。然而，SRM方法受到单次采集过程中可成像的合适荧光团数量的限制，以及抗体洗涤和重新染色以进行有机染料多重标记过程中抗原的损失。我们报告了使用DNA辅助单分子定位显微镜（SMLM）在350 - 400 nm厚的神经元组织切片的突触前和突触后可视化多个蛋白质靶点。在单个标记步骤中，与短DNA寡核苷酸偶联的抗体通过依次交换成像缓冲液中存在的荧光团标记的互补寡核苷酸来可视化多个靶点。这种方法避免了使用多轮免疫标记时对结构完整性的潜在影响，并消除了色差，因为所有靶点都使用单一激发激光波长进行成像。该方法在横向分辨率优于25 nm的半薄组织切片中对多靶点成像表现出稳健性，为单分子SRM的结构细胞生物学研究铺平了道路。
