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 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 13C atoms originating from 13C-labeled glucose can be exploited by MIP-FISH to discriminate and identify 13C-labeled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure- function analyses for microbiology.

Automated summary

A mid-infrared photothermal-fluorescence in situ hybridization (MIP-FISH) platform is developed for simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput. By improving MIP imaging, isotopically labeled compounds incorporated into proteins of individual bacterial cells can be sensitively detected, while FISH labeling with rRNA-targeted probes enables the identification of the analyzed cells. In proof-of-concept experiments, MIP-FISH successfully discriminates and identifies labeled bacterial cells within a complex human gut microbiome sample based on the clear spectral red shift in the protein amide I region caused by incorporation of 13C atoms from 13C-labeled glucose. This study opens up new opportunities for single-cell structure-function analyses in microbiology.