Mapping cellular metabolism in tissues: multi-parameter assays combining live cell fluorescence microscopy and islet/tissue-on-a-chip.

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Cells sense and adapt to their microenvironment through changes in metabolism. However, these changes cannot be studied using metabolomic assays alone as they are often transient and occur within only a fraction of the cell population being studied at a given time. To fully understand cell sensing and adaption, particularly within the context of tissue, methods are needed to measure live cell metabolic responses with high temporal and spatial resolution. The Rocheleau Lab has long-standing interest and expertise in combining live cell fluorescence imaging (fluorescent protein sensors) with microfluidic device manipulation of living tissue (specifically, pancreatic islets). Pancreatic islets are a model system of glucose-stimulated insulin secretion that further affords the opportunity to study fundamental mechanisms of cellular plasticity, survival, and proliferation within a tissue. With the support of NSERC funding over the last 6 years, we have (i) developed fluorescent protein sensors based on fluorescence anisotropy imaging (termed Apollo) to measure organelle NADPH redox state and ER stress, indicators of cellular metabolism, (ii) developed methods to dynamically image O2-consumption and insulin secretion from individual islets, indicators of cellular physiology, and (iii) designed microfluidic devices to translate precision-cut pancreas slices (slice-on-a-chip) to multiple live cell imaging platforms (confocal and light sheet). Critically, each of these assays are spectrally tunable and thus amenable for simultaneous measurements on individual live samples. Our long-term objective is to design new Apollo sensors and on-chip-assays, with the goal of combining these methods to address mechanisms of glucose-stimulated insulin secretion from pancreatic islets. These studies propose three short-term objectives: 1)Develop new fluorescent protein Apollo sensors while exploring strategies to improve sensor dynamic range. 2)Develop islet-on-a-chip microfluidic assays to concurrently measure O2-consumption rate, insulin secretion, and glucagon secretion from individual isles with high temporal resolution. 3)Design microfluidic devices to image the metabolism and hormone secretion of islets within precision cut pancreas tissue slices (slice-on-a-chip). This research program critically relies on our multidisciplinary expertise in fluorescent proteins, microfluidic devices, and quantitative live cell fluorescence microscopy. Our focus on islet biology will reveal ground-breaking insight into metabolism secretion coupling with easy translation to clinical application and the study of other tissues. Finally, this research program will provide HQP with advanced training in islet and beta-cell biology, quantitative fluorescence microscopy, bioengineering, microfluidic device design, biophysics, and research design & dissemination, all skills at the forefront of modern research and highly sought in academic and industrial settings.