Mid-Infrared Photothermal–Fluorescence In Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells
Yeran Bai, Zhongyue Guo, Fátima C. Pereira, Michael Wagner, Ji‐Xin Cheng
Abstract
High Resolution Image Download MS PowerPoint Slide Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput are necessary to answer the question of “who eats what, when, and where” in complex microbial communities. Here, we present a mid-infrared photothermal–fluorescence in situ hybridization (MIP–FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically labeled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labeling with rRNA-targeted probes enabled the identification of the analyzed cells. In proof-of-concept experiments, we showed that the clear spectral red shift in the protein amide I region due to incorporation of 13 C atoms originating from 13 C-labeled glucose can be exploited by MIP–FISH to discriminate and identify 13 C-labeled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure–function analyses for microbiology.