An integrative approach for imaging and quantitative analysis of gut microbiota growth <i>in vivo</i> using fluorescent D-amino acid labeling and fluorescence <i>in situ</i> hybridization

The profound influence of gut microbiota on human health has been well-recognized; however, substantial gaps remain in our understanding of the highly diverse and dynamic processes of microbial growth and activities in the gut. Conventional methods, which primarily rely on DNA sequencing, provide limited insights into these aspects. This paper presents a protocol that integrates fluorescent D-amino acid (FDAA) metabolic labeling with fluorescence <i>in situ</i> hybridization (FISH) for imaging and quantitatively analyzing the <i>in vivo</i> growth of gut microbiota. By administering two FDAAs sequentially through mouse gavage, we label the peptidoglycan of gut bacteria in their native environment, allowing the labeling signals on bacterial cell walls to serve as markers of cellular proliferation and division. We have also demonstrated that the intensity of FDAA labeling directly correlates with the metabolic activity of gut bacteria. Additionally, FISH is employed to distinguish specific bacterial taxa of interest via fluorescence microscopy or flow cytometry. This integrative method greatly enhances our capacity to visualize and measure the <i>in vivo</i> growth and metabolic states of various gut bacteria, thereby illuminating the previously obscured "dark matter" in the gut ecosystem.