Stable isotope labeling of microbial taxa of interest and their sorting provide an efficient and direct way to answer the question “who does what?” in complex microbial communities when coupled with fluorescence in situ hybridization or downstream ‘omics’ analyses. We have developed a platform for automated Raman-based sorting in which optical tweezers and microfluidics are used to sort individual cells of interest from microbial communities on the basis of their Raman spectra. This sorting of cells and their downstream DNA analysis, such as by mini-metagenomics or single-cell genomics, or cultivation permits a direct link to be made between the metabolic roles and the genomes of microbial cells within complex microbial communities, as well as targeted isolation of novel microbes with a specific physiology of interest. We describe a protocol from sample preparation through Raman-activated live cell sorting. Subsequent cultivation of sorted cells is described, whereas downstream DNA analysis involves well-established approaches with abundant methods available in the literature. Compared with manual sorting, this technique provides a substantially higher throughput (up to 500 cells per h). Furthermore, the platform has very high sorting accuracy (98.3 ± 1.7%) and is fully automated, thus avoiding user biases that might accompany manual sorting. We anticipate that this protocol will empower in particular environmental and host-associated microbiome research with a versatile tool to elucidate the metabolic contributions of microbial taxa within their complex communities. After a 1-d preparation of cells, sorting takes on the order of 4 h, depending on the number of cells required.

感兴趣的微生物类群的稳定同位素标记及其分类为回答“谁做什么？”这个问题提供了一种有效而直接的方法。当与荧光原位杂交或下游“组学”分析相结合时，在复杂的微生物群落中。我们开发了一个基于拉曼光谱的自动分选平台，其中光镊和微流体用于根据拉曼光谱从微生物群落中分选感兴趣的单个细胞。这种细胞分类及其下游 DNA 分析，例如通过微型宏基因组学或单细胞基因组学，或培养允许在代谢作用和复杂微生物群落中微生物细胞的基因组之间建立直接联系，以及靶向分离具有特定生理学的新型微生物。我们描述了从样品制备到拉曼活化活细胞分选的协议。描述了分选细胞的后续培养，而下游 DNA 分析涉及成熟的方法和文献中可用的丰富方法。与手动分选相比，该技术提供了更高的吞吐量（每小时高达 500 个细胞）。此外，该平台具有非常高的分拣精度（98.3 ± 1.7%）并且是完全自动化的，从而避免了可能伴随手动分拣的用户偏见。我们预计该协议将赋予特别是环境和宿主相关微生物组研究的能力，并提供一种多功能工具来阐明微生物类群在其复杂群落中的代谢贡献。在细胞的 1 d 准备后，分选大约需要 4 小时，