Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identify four critical sample preparation factors contributing to the method’s sensitivity. We validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane protein distribution. We evaluate the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale MYO15A-L rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM extends the application of SEM-based nanoscale protein localization to the detection of intracellular epitopes on the exposed surfaces of any cell.

电子显微镜与免疫金标记相结合是蛋白质定位最精确的工具。然而，这些方法要么很麻烦，导致样本数量小且定量有限，要么仅限于识别膜外部的蛋白质表位。在这里，我们介绍 SUB-immunogold-SEM，这是一种扫描电子显微镜技术，可检测膜附近的细胞内蛋白质表位。我们确定了影响该方法灵敏度的四个关键样品制备因素。我们通过细胞骨架和跨膜蛋白分布的精确定位和高效量化来验证其功效。我们评估了 SUB-immunogold-SEM 对具有高度分化顶端表面的细胞的能力：（i）听觉毛细胞，揭示了静纤毛顶端存在纳米级 MYO15A-L 环； (ii) 呼吸道多纤毛细胞，绘制 SARS-CoV-2 受体 ACE2 沿运动纤毛的分布图。 SUB-immunogold-SEM 将基于 SEM 的纳米级蛋白质定位的应用扩展到任何细胞暴露表面上的细胞内表位的检测。