Understanding the nano-architecture of protein machines in diverse subcellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While single-molecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS³TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D direct STORM (dSTORM) imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 targets in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision, allowing us to define distinct nano-architectural features of protein distribution within the presynaptic nerve terminal.

尽管在超分辨率显微镜领域取得了快速进展，但了解蛋白质在不同亚细胞区室中的纳米结构仍然是一个挑战。虽然单分子定位显微镜技术可以以近乎分子的分辨率可视化和识别细胞结构，但同一样品中数十种靶蛋白的多重标记尚未得到常规实现。但是，单样本多路复用对于检测有可能在多样本平均中丢失的模式至关重要。在这里，我们报告了maS ³ TORM（多路自动连续染色随机光学重建显微镜），这是一种能够实现全自动3D直接STORM（dSTORM）成像和溶液交换，采用重染色方案以实现单个生物样品中高度复用的蛋白质定位。我们展示了单培养细胞中15个靶标和单个神经元组织样品中16个靶标的3D超分辨率图像，定位精度小于10 nm，这使我们能够定义突触前神经末梢中蛋白质分布的独特纳米结构特征。