Autofluorescence lifetime microscopy for label-free detection of cell metabolism for cell biology research

项目摘要/摘要 这是由早期阶段提交的最大化调查者研究奖(MIRA)的申请 ESI调查员亚历克斯·沃尔什博士。沃尔什博士的研究项目侧重于定性和 用于细胞代谢和线粒体定量的自体荧光寿命显微镜的发展 活细胞的功能。细胞代谢异常和线粒体功能障碍是许多疾病的标志。 以及病态。此外，许多药物和药剂，包括麻醉，或者 有直接的作用机制，通过改变新陈代谢信号或由于受损而产生间接毒性 线粒体的功能。目前评估细胞新陈代谢的研究方法包括基于细胞的分析，如 基因组、代谢和耗氧量分析以及全身成像分析，例如 氟脱氧葡萄糖正电子发射断层扫描和~(13)C磁共振成像。然而， 这些现有的工具具有有限的空间和时间分辨率以及分析的标签依赖性质 防止评估动态代谢状态和同一样本的多个纵向评估。 线粒体内还原型烟酰胺腺嘌呤二核苷酸(NADH)的自体荧光寿命成像 线粒体内的胞浆和黄素腺嘌呤二核苷酸(FAD)呈现一种独特的、无标记的、高度... 评价细胞新陈代谢的分解技术。自体荧光寿命显微镜不依赖于 化学或抗体标记，是非接触式的，广泛适用于任何细胞或组织，非常适合纵向 研究，并与二次化验兼容。然而，目前采用自体荧光寿命 受制于设计和执行荧光寿命实验所需的高级专业知识，默默无闻 在荧光寿命度量和分子特异性之间的相关性方面，以及缺乏可靠的模型来 根据自发荧光特征确定代谢表型和线粒体功能。因此，Dr。 沃尔什未来五年的目标将解决这些限制。目标1：确定敏感性和 自体荧光寿命成像识别细胞代谢状态和途径利用的特异性。目标 2：量化常见实验室模型中的自体荧光寿命成像特征以确定稳健性 用于报告细胞新陈代谢的自发荧光测量方法。目标3：开发、测试和优化自发荧光 用于量化线粒体动力学和功能的成像方案。这项提案的结果将使 用高时间分辨率和高特异性的代谢测量来评估活着的细胞代谢 细胞。此外，自发荧光成像将提供一个平台，以评估药物和 药物，包括麻醉剂，对活细胞的细胞新陈代谢和线粒体功能， 组织和活体内。

PROJECT SUMMARY/ABSTRACT This is an application for a Maximizing Investigator’s Research Award (MIRA) submitted by the Early Stage Investigator (ESI), Dr. Alex Walsh. Dr. Walsh’s research program focuses on the characterization and development of autofluorescence lifetime microscopy for the quantification of cell metabolism and mitochondria function of living cells. Abnormal cell metabolism and mitochondria dysfunction is a hallmark of many diseases and pathological states. Furthermore, many drugs and pharmacological agents, including anesthesia, either have direct mechanisms of action through altered metabolism signaling or indirect toxicities due to impaired mitochondria function. Current research assays to evaluate cellular metabolism include cell-based assays such genomic, metabolomic, and oxygen-consumption assays and whole-body imaging assays such as fluorodeoxyglucose-positron emission tomography and carbon-13 magnetic resonance imaging. However, these existing tools have limited spatial and temporal resolutions and the label-dependent nature of the assays prevents assessment of dynamic metabolic states and multiple longitudinal assessments of the same samples. Autofluorescence lifetime imaging of reduced nicotinamide adenine dinucleotide (NADH) within the mitochondria and cytosol and flavin adenine dinucleotide (FAD) within mitochondria presents a unique, label-free, high- resolution technique to evaluate cell metabolism. Autofluorescence lifetime microscopy is not dependent on chemical or antibody labels, is non-contact, broadly applicable to any cell or tissue, well-suited for longitudinal studies, and compatible with secondary assays. However, adoption of autofluorescence lifetime is currently hindered by the advanced expertise needed to design and perform fluorescence lifetime experiments, obscurity in the correlation between fluorescence lifetime metrics and molecular specificity, and a lack of robust models to determine metabolic phenotype and mitochondria function from autofluorescence features. Therefore, Dr. Walsh’s goals for the next five years will address these limitations. Goal 1: Determine the sensitivity and specificity of autofluorescence lifetime imaging to identify cellular metabolic states and pathway utilization. Goal 2: Quantify autofluorescence lifetime imaging features across common lab models to determine the robustness of autofluorescence metrics to report cellular metabolism. Goal 3: Develop, test, and optimize autofluorescence imaging protocols to quantify mitochondria dynamics and function. The outcomes of this proposal will enable metabolic measurements with high temporal resolution and specificity to evaluate cellular metabolism in living cells. Additionally, autofluorescence imaging will provide a platform to evaluate the impacts of drug and pharmacological agents, including anesthetics, on cell metabolism and mitochondria function in living cells, tissues, and in vivo.