The vast majority of environmental bacteria remain uncultured, despite two centuries of effort in cultivating microorganisms. Our knowledge of their physiology and metabolic activity depends to a large extent on methods capable of analyzing single cells. Bacterial identification is a key step required by all currently used single-cell imaging techniques and is typically performed by means of fluorescent labeling. However, fluorescent cells cannot be visualized by ion- and electron microscopy and thus only correlative, indirect, cell identification is possible. Here we present a new method of bacterial identification by in situ hybridization coupled to the deposition of elemental silver nanoparticles (silver-DISH). We show that hybridized cells containing silver can be directly visualized by light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, secondary ion mass spectrometry (nanoSIMS), and confocal Raman micro-spectroscopy. Silver-DISH did not alter the isotopic (¹³C) and elemental composition of stable-isotope probed cells more than other available hybridization methods, making silver-DISH suitable for broad applications in stable-isotope labeling studies. Additionally, we demonstrate that silver-DISH can induce a surface-enhanced Raman scattering (SERS) effect, amplifying the Raman signal of biomolecules inside bacterial cells. This makes silver-DISH the only currently available method that is capable of delivering a SERS-active substrate inside specifically targeted microbial cells.

尽管在微生物培养上进行了两个世纪的努力，但绝大多数环境细菌仍未进行培养。我们对它们的生理和代谢活性的了解在很大程度上取决于能够分析单个细胞的方法。细菌鉴定是所有当前使用的单细胞成像技术所需的关键步骤，通常是通过荧光标记进行的。但是，荧光细胞不能通过离子显微镜和电子显微镜观察到，因此只能进行相关的间接细胞鉴定。在这里，我们提出了一种通过原位细菌鉴定的新方法杂交与元素银纳米颗粒的沉积偶联（silver-DISH）。我们显示，可以通过光学显微镜，扫描电子显微镜，能量色散X射线光谱，二次离子质谱（nanoSIMS）和共聚焦拉曼光谱直接观察包含银的杂交细胞。Silver-DISH不会改变同位素（¹³C）稳定同位素探测的细胞的元素组成比其他可用的杂交方法更多，这使得Silver-DISH适合在稳定同位素标记研究中广泛应用。此外，我们证明银-DISH可以诱导表面增强的拉曼散射（SERS）效应，从而放大细菌细胞内生物分子的拉曼信号。这使Silver-DISH成为当前唯一能够在特定目标微生物细胞内传递SERS活性底物的方法。