Illuminating the spatial and temporal diversity of the gastrointestinal microbial ecosystem with label-free nonlinear optical microscopy

PROJECT SUMMARY/ABSTRACT Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal condition that affects an estimated 2.4-3.1 million people in the US. IBD is increasing in prevalence and cost, and linked to the development of colorectal cancer. While recent progress has improved our understanding of IBD pathophysiology, many unknowns remain about the relationship between host and microbe interactions in the intestinal microenvironment. A stronger understanding of the microscopic biochemical basis of IBD could lead more effective prevention and treatment. Established technologies lack the ability to observe bacteria and host cells with sufficient temporal and spatial resolution, with most methods performing a bulk analysis ex vivo. Many established imaging methods do not have sufficient spatial resolution to observe bacteria cells, and established super-resolution imaging methods are not well-suited for dynamic imaging in vivo. IBD is one of many driving forces that has led to a critical need for new methods to observe and quantify the intestinal microenvironment with high spatial and temporal resolution. This proposal seeks to address this need by developing and testing a novel optical imaging technology to observe the dynamics and heterogeneity of host-microbe interactions in the live mouse intestinal microenvironment. A new method for multiphoton super-resolution microscopy will be developed, compatible with label-free autofluorescence imaging of NAD(P)H, FAD, and tryptophan, all of which are essential to host and microbe metabolism in the intestine. Furthermore, this method will be used for super-resolution imaging of label-free biochemical information with hyperspectral coherent Raman scattering microscopy and structural information with sum-frequency generation of collagen. In order to accelerate the acquisition of 5D data with temporal and spectral information (x-y-z-t-λ), computational methods will be employed to acquire and save only the essential information, enabling imaging fast dynamics and large volumes of data. These newly established methods will be used to image and characterize the microenvironment of the large intestines of mice in vivo, examining differences between IBD and control mice, and metabolic changes associated with different grades of IBD. This project fulfills a critical need for new approaches to study the metabolism of the intestinal microenvironment in vivo with unprecedented spatial and temporal resolution. While focused on IBD, this technology has broad potential for studying other complex tissue microenvironments, and will be especially useful for bringing the next generation of microscopy methods to microbiology.