The gut microbiome has a considerable impact on human health. Furthermore, it is very sensitive to the in vivo environment changes. Entacapone (ENT) is one important human-targeted drug for the treatment of Parkinson’s disease shown to largely impact the growth of individual organisms in the gut microbiome. To resolve ENT incorporation and impact on gut microbiome activity in situ, we employed stimulated Raman scattering - fluorescence in situ hybridization (SRS-FISH) along with ultrafast delay line tuning. The hyperspectral chemical images acquired with this method could better preserve and separate the chemical information, thus enabled high-throughput identity-specific single cell metabolism analysis and mapping of drug incorporation by individual microbiome cells.
We developed a mid-infrared photothermal imaging (MIPI) - fluorescence in-situ hybridization (FISH) microscope that enables simultaneous identity probing and metabolic activity imaging for bacteria in a complex environment. We added the 13C-glucose in the bacteria culture medium and monitored the newly synthesized protein composition by MIPI at amide I band. Bacterial taxa were targeted with fluorescently-labelled FISH probes and imaged with the widefield epi-fluorescence imaging that built on to the MIP microscope. The sub-micrometer spatial resolution of MIPI enables the characterization at single bacterium level. The proposed platform provides the link between genotype and phenotype and would be a powerful tool to deepen our understanding of microbiome.
For high-throughput link of microbiome function and taxonomic identity at the single cell level, we established a stimulated Raman scattering (SRS)-fluorescence in situ hybridization (FISH) platform. SRS combined with the deuterium-based isotope probing enables chemical mapping and reveals metabolic activity of bacteria. Fluorescently tagged oligonucleotide probes identify different bacteria and are detected through two photon fluorescence (TPF) microscopy. As a proof-of-principle demonstration, we tested the platform in a mixture of two distinct gut microbiota taxa with different deuterium labeling levels. This established platform not only provides enormous potential to study microbiota in the complex environment, but also the simultaneous observation of phenotype and genotype in the general biological systems.
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