Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Celsiustrasse 1, 28359, Bremen, Germany; Institute of Biogeochemistry and Pollutant Dynamics, Department of Environmental Systems Science, Swiss Federal Institute of Technology Zurich, Universitätsstrasse 16, 8092 Zurich, Switzerland.
Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Celsiustrasse 1, 28359, Bremen, Germany.
Syst Appl Microbiol. 2020 May;43(3):126086. doi: 10.1016/j.syapm.2020.126086. Epub 2020 Apr 27.
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 (C) 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.
尽管两个世纪以来一直致力于培养微生物，但绝大多数环境细菌仍然未被培养。我们对它们的生理学和代谢活性的了解在很大程度上依赖于能够分析单细胞的方法。细菌鉴定是所有当前使用的单细胞成像技术都需要的关键步骤，通常通过荧光标记来完成。然而，离子和电子显微镜无法观察到荧光细胞，因此只能进行相关的、间接的细胞鉴定。在这里，我们提出了一种通过原位杂交结合元素银纳米颗粒（银-DISH）沉积来鉴定细菌的新方法。我们表明，含有银的杂交细胞可以通过明场显微镜、扫描电子显微镜、能量色散 X 射线光谱、二次离子质谱（nanoSIMS）和共聚焦拉曼微光谱直接观察到。与其他可用的杂交方法相比，银-DISH 不会改变同位素（C）和稳定同位素探测细胞的元素组成，这使得银-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 可以诱导表面增强拉曼散射（SERS）效应，放大细菌细胞内生物分子的拉曼信号。这使得银-DISH 成为目前唯一能够将 SERS 活性底物递送到特定靶向微生物细胞内的方法。
