Examining cellular metabolism using two-photon and confocal microscopy of NAD(P)H and flavin autofluorescence

细胞代谢营养物质以产生和储存生命能量。 重要的是，细胞代谢是动态的，并适应营养的可用性，以生存。 研究新陈代谢的经典方法是在试管中磨碎细胞，测量生化成分。 然而，这种方法需要大量的细胞，并且不能在同一样品上重复收集，因为它是终末事件。 我们的研究计划利用了一种替代方法，该方法使用每个活细胞的固有特性：代谢的变化改变了细胞的固有荧光特性（自发荧光）。 我们利用自发荧光和代谢之间的这种联系来了解细胞如何响应和适应各种治疗。 为了测量小鼠组织中细胞的自发荧光，我们使用带有各种不同颜色激光的显微镜。 重要的是，我们可以重复测量单个样品的自发荧光，这使我们能够测量一段时间内对特定治疗的反应。 在第一项研究中，我们使用自体荧光显微镜来确定细胞代谢如何响应缺氧而变化。 在第二项研究中，我们使用其他可用的光学传感器与自体荧光进行比较，并进一步检查代谢。 第三项研究开发了一种新型传感器，用于收集多种代谢过程的特定比较数据。 这些研究将增加我们对细胞代谢如何响应环境变化的理解，并导致新的工具来同时动态地测量代谢的各个方面。

Cells metabolize nutrients to generate and store energy for life. Critically, cell metabolism is dynamic and adapts to nutrient availability in order to survive. Metabolism has classically been studied by grinding-up cells to measure biochemical composition in a test-tube. However, this approach requires large numbers of cells and cannot be collected repetitively on the same sample since it is a terminal event. Our research program takes advantage of an alternative approach that uses an inherent property of every living cell: a change in metabolism modifies the inherent fluorescent properties of a cell (autofluorescence). We use this link between autofluorescence and metabolism to understand how cells respond and adapt to various treatments. To measure autofluorescence from cells in a mouse tissue, we use a microscope with a variety of different coloured lasers. Importantly, we can repetitively measure the autofluorescence from a single sample, which allows us to measure the response to a specific treatment over a period of time. In the first proposed study, we use autofluorescence microscopy to determine how cell metabolism changes in response to the deprivation of oxygen. In the second study, we use other available optical sensors to compare with autofluorescence and further examine metabolism. The third study develops a novel sensor to gather specific comparative data on multiple processes of metabolism. These studies will increase our understanding of how cellular metabolism changes in response to the surroundings and lead to new tools to simultaneously and dynamically measure various aspects of metabolism.