Quantifying mucosal hemodynamics in a murine model of Ulcerative Colitis with diffuse reflectance spectroscopy

Ulcerative colitis (UC) is a gastrointestinal, autoimmune disease that causes ulceration and inflammation of the colon with an incidence 10 out of every 100,000 people in North America and Western Europe. Though the exact etiology is uncertain, a number of studies have shown that inflammatory cells along with environmental factors, genetics, and lifestyle habits can contribute to the sustained inflammatory response. In order to determine the cellular mechanism behind relapse and remission of UC, researchers have frequently employed immunohistochemistry, western blotting and gene sequencing, but these destructive analysis methods require the removal of a sample, necessarily limiting these methods to non-living tissues. There is an emerging interest in using non-invasive techniques to study the in vivo, longitudinal effects of UC on the mucosa in the colon. Here we have developed a mouse model of UC using dextran sulfate sodium and a non-invasive spectroscopy monitoring modality to study the changes in the tissue hemodynamics during active UC

Live-Cell Imaging Quantifies Changes in Function and Metabolic NADH Autofluorescence During Macrophage-Mediated Phagocytosis of Tumor Cells

The immune system has evolved to detect foreign antigens and deliver coordinated responses, while minimizing “friendly fire.” Until recently, studies investigating the behavior of immune cells were limited to static in vitro measurements. Although static measurements allow for real-time imaging, results are often difficult to translate to an in vivo setting. Multiphoton microscopy is an emerging method to capture spatial information on subcellular events and characterize the local microenvironment. Previous studies have shown that multiphoton microscopy can monitor changes in single-cell macrophage heterogeneity during differentiation. Therefore, there is a need to use multiphoton microscopy to monitor molecular interactions during immunological activities like phagocytosis. Here we investigate the correlation between phagocytic function and changes in endogenous optical reporters during phagocytosis. In vitro autofluorescence imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) was used to detect metabolic changes in macrophages during phagocytosis. More specifically, optical redox ratio, mean NADH fluorescence lifetime and ratio of free to protein-bound NADH were used to quantify changes in metabolism. Results show that IFN-γ (M1) macrophages showed decreased optical redox ratios and mean NADH lifetime while phagocytosing immunogenic cancer cells compared to metastatic cells. To validate phagocytic function, a fluorescence microscopy-based protocol using a pH-sensitive fluorescent probe was used. Results indicate that M0 and M1 macrophages show similar trends in phagocytic potential. Overall, this work demonstrates that in vitro multiphoton imaging can be used to longitudinally track changes in phagocytosis and endogenous metabolic cofactors.</p