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

细胞通过代谢的变化来感知和适应微环境。然而，这些变化不能单独使用代谢组学分析来研究，因为它们通常是短暂的，并且在给定时间内仅发生在正在研究的细胞群的一小部分中。为了充分了解细胞的感知和适应，特别是在组织的背景下，需要高时间和空间分辨率的方法来测量活细胞代谢反应。Rocheleau实验室长期致力于将活细胞荧光成像（荧光蛋白传感器）与活组织（特别是胰岛）的微流体装置操作相结合。胰岛是葡萄糖刺激胰岛素分泌的模型系统，它进一步为研究组织内细胞可塑性、存活和增殖的基本机制提供了机会。在NSERC过去6年的资助下，我们已经(i)开发了基于荧光各向异性成像的荧光蛋白传感器（称为Apollo），用于测量细胞器NADPH氧化还原状态和内质网应激，细胞代谢指标；（ii）开发了动态成像单个胰岛的o2消耗和胰岛素分泌的方法，细胞生理学指标；（iii）设计了微流体装置，将精确切割的胰腺切片（片上切片）转化为多个活细胞成像平台（共聚焦和光片）。至关重要的是，这些分析中的每一个都是光谱可调的，因此可以同时测量单个活样品。我们的长期目标是设计新的阿波罗传感器和芯片上检测，目标是结合这些方法来解决胰岛葡萄糖刺激胰岛素分泌的机制。这些研究提出了三个短期目标：1)开发新的荧光蛋白阿波罗传感器，同时探索提高传感器动态范围的策略。2)发展胰岛芯片微流控技术，以高时间分辨率同时测量单个胰岛的臭氧消耗率、胰岛素分泌和胰高血糖素分泌。3)设计微流控装置，在精确切割的胰腺组织切片（切片-芯片）中成像胰岛的代谢和激素分泌。该研究项目主要依赖于我们在荧光蛋白、微流体装置和定量活细胞荧光显微镜方面的多学科专业知识。我们对胰岛生物学的关注将揭示代谢分泌耦合的突破性见解，并易于转化为临床应用和其他组织的研究。最后，该研究项目将为HQP提供胰岛和β细胞生物学、定量荧光显微镜、生物工程、微流体装置设计、生物物理学、研究设计和传播等方面的高级培训，所有这些技能都处于现代研究的前沿，在学术和工业环境中备受追捧。

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.