Three-dimensional imaging for the quantification of spatial patterns in microbiota of the intestinal mucosa

Abstract

Significance Many human diseases are causally linked to the gut microbiota, yet the field still lacks mechanistic understanding of the underlying complex interactions, because existing tools cannot simultaneously quantify microbial communities and their native context. In this work, we provide an approach to tissue clearing and preservation that enables 3D visualization of the biogeography of the host–microbiota interface. We combine this tool with sequencing and multiplexed microbial labeling to provide the field with a platform on which to discover patterns in the spatial distribution of microbes. We validated this platform by quantifying bacterial distribution in cecal mucosa at different stages of antibiotic exposure. This approach may enable researchers to formulate and test new hypotheses about host–microbe and microbe–microbe interactions.

Quantitative whole-tissue 3D imaging reveals bacteria in close association with mouse jejunum mucosa

“…Here, we leveraged whole-tissue 3D imaging tools [34] that allowed us to map both microbe-microbe and host-microbe spatial organization at the mucosal interface over large areas and in 3D. For example, leveraging a protective surface gel [34], we successfully retained large surface aggregates and bacteria, which can otherwise be easily lost during sample processing and evade detection. Furthermore, we visualized in 3D how bacterial clusters connect in between and wrap around the villi.…”

“…Using a mouse model of environmental enteropathy and building on our whole-tissue clearing and 3D-imaging tools [34], we documented dense bacterial association with the jejunum mucosa, suggestive of colonization. We anticipated that bacterial colonization of jejunum mucosa would be rare across space and over time in healthy organisms, therefore, we leveraged three strategies.…”

“…Furthermore, mucosal colonizers should appear as multicellular clusters rather than individual cells, indicative of cellular division and growth in the mucosa. Here, we leveraged whole-tissue 3D imaging tools [34] that allowed us to map both microbe-microbe and host-microbe spatial organization at the mucosal interface over large areas and in 3D. For example, leveraging a protective surface gel [34], we successfully retained large surface aggregates and bacteria, which can otherwise be easily lost during sample processing and evade detection.…”

“…We used our 3D imaging tools [34] to map the exact bacterial location with respect to the complex mucosal landscape of the host and look for evidence of bacterial adhesion to the mucosa. First, to evaluate imaging workflow, we performed a pilot experiment by taking one empty jejunum tissue sample from each malnourished group through the whole-tissue 3D imaging pipeline (Fig.…”

“…CUBIC has been used to visualize host-microbe interface in 3D along the entire gut of mice, however, this method did not allow to image native bacteria with taxonomic resolution [33]. Finally, we have recently developed CLARITY-based imaging tools that allow one to scan large areas of intestinal mucosa and profile heterogeneous bacterial association with intestinal mucosa in 3D with highly sensitive and specific taxonomic resolution of bacteria [34]. Thus, we reasoned that our tools are suited to study rare events of bacterial colonization of SI mucosa.…”