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

细胞通过新陈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分析来研究，因为它们通常是瞬时的，在给定的时间只发生在被研究的细胞群体的一小部分中。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的细胞代谢反应。罗切洛实验室在将活细胞荧光成像(荧光蛋白传感器)与微流控设备操作活组织(特别是胰岛)相结合方面有着长期的兴趣和专业知识。胰岛是葡萄糖刺激的胰岛素分泌的模型系统，进一步提供了研究组织内细胞可塑性、存活和增殖的基本机制的机会。在NSERC过去6年的资助下，我们(I)开发了基于荧光各向异性成像(称为Apollo)的荧光蛋白质传感器来测量细胞器NADPH氧化还原状态和内质网应激，这是细胞代谢的指标，(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，以及(Iii)设计了微流体设备，可以将精密切割的胰腺切片(芯片上切片)转换到多个活细胞成像平台(共焦和光片)。关键的是，这些分析中的每一种都是光谱可调的，因此可以在单独的活体样品上同时测量。我们的长期目标是设计新的阿波罗传感器和芯片上测试，目标是将这些方法结合起来，研究葡萄糖刺激的胰岛胰岛素分泌机制。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)发展芯片上的胰岛微流控技术，以高时间分辨率同时测量单个胰岛的耗氧率、胰岛素分泌和胰升糖素的分泌。3)设计微流控装置，对精密切割的胰腺组织切片(切片)内的胰岛代谢和激素分泌进行成像。这一研究项目在很大程度上依赖于我们在荧光蛋白、微流体设备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对新陈代谢分泌的开创性见解，并易于转化为临床应用和其他组织的研究。最后，这项研究计划将为HQP提供胰岛和β细胞生物学、定量荧光显微镜、生物工程、微流体设备设计、生物物理学以及研究设计和传播方面的高级培训，所有这些技能都处于现代研究的前沿，在学术界和工业界备受追捧。

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.