Mid-Infrared Photothermal-Fluorescence in Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells
Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput is 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-labelled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labelling 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-labelled-glucose can be exploited by MIP-FISH to discriminate and identify $^{13}$C-labelled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure-function analyses for microbiology.
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