Genetically encoded tags for single-molecule imaging in electron microscopy (EM) are long-awaited. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated tags. Next, we exploited this mechanism to develop approaches for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies and mitochondrial matrices. We further implemented cysteine-rich tag–antibody fusion proteins as new immuno-EM probes. Thus, our approaches should allow biologists to address a wide range of biological questions at the single-molecule level in cellular ultrastructural contexts.

期待用于电子显微镜（EM）中单分子成像的遗传编码标签。在这里，我们报告了一种方法，可以直接在富含半胱氨酸的标签上直接合成EM可见的金纳米颗粒（AuNPs），用于细胞中的单分子可视化。我们首先发现了一种自动成核抑制机制，该机制允许在分离的标签上特异性合成AuNP。接下来，我们利用这种机制开发了用于原核细胞中蛋白质单分子检测的方法，并实现了前所未有的标记效率。然后，我们将其扩展到更复杂的真核细胞，并成功检测了针对各种细胞器的蛋白质，包括内质网（ER）和核包膜，ER内腔，核孔，纺锤极体和线粒体基质。我们进一步实现了富含半胱氨酸的标签抗体融合蛋白作为新的免疫电磁探针。因此，我们的方法应允许生物学家在细胞超微结构背景下以单分子水平解决广泛的生物学问题。