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

用于细胞生物学研究的细胞代谢无标记检测的自体荧光寿命显微镜

• 批准号: 10276463
• 负责人: Alexandra Walsh
• 金额: $ 36.12万
• 依托单位: TEXAS ENGINEERING EXPERIMENT STATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-23 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10276463
• 关键词: Abnormal Cell; Address; Adoption; Anesthesia procedures; Anesthetics; Antibodies; Award; Biological Assay; Carbon; Cells; Cellular Metabolic Process; Cellular biology; Chemicals; Clinical Research; Cytosol; Defect; Detection; Development; Disease; Ensure; Flavin-Adenine Dinucleotide; Fluorescence; Genomics; Goals; Image; Impairment; Label; Longitudinal Studies; Magnetic Resonance Imaging; Measurement; Metabolic; Metabolic Diseases; Metabolism; Microscopy; Mitochondria; Modeling; Molecular; Nature; Neurodegenerative Disorders; Nicotinamide adenine dinucleotide; Outcome; Oxygen Consumption; Pathologic; Pathway interactions; Patient Care; Pharmaceutical Preparations; Pharmacology; Protocols documentation; Reporting; Research; Research Personnel; Resolution; Sampling; Sensitivity and Specificity; Signal Transduction; Specificity; Techniques; Testing; Tissues; Toxic effect; base; design; experimental study; fluorodeoxyglucose positron emission tomography; in vivo; insight; metabolic abnormality assessment; metabolic phenotype; metabolomics; mitochondrial dysfunction; mitochondrial metabolism; preclinical study; prevent; programs; side effect; temporal measurement; tool; whole body imaging

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.

项目总结/摘要 这是一个申请最大化研究者的研究奖（MIRA）提交的早期阶段 研究员（ESI），Alex沃尔什博士。沃尔什博士的研究计划侧重于表征和 用于定量细胞代谢和线粒体的自体荧光寿命显微术的发展 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 和病理状态。此外，许多药物和药理学试剂，包括麻醉剂， 通过改变代谢信号传导或由于受损的代谢信号传导而产生间接毒性， 线粒体功能目前用于评估细胞代谢的研究测定包括基于细胞的测定，例如 基因组学、代谢组学和耗氧量测定以及全身成像测定， 氟脱氧葡萄糖-正电子发射断层扫描和碳-13磁共振成像。然而，在这方面， 这些现有的工具具有有限的空间和时间分辨率 防止动态代谢状态的评估和相同样品的多个纵向评估。 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 线粒体内的细胞质和黄素腺嘌呤二核苷酸（FAD）呈现出独特的、无标记的、高水平的 分辨率技术来评估细胞代谢。自体荧光寿命显微镜不依赖于 化学或抗体标记，是非接触的，广泛适用于任何细胞或组织，非常适合纵向 研究，并与二次检测兼容。然而，目前采用自发荧光寿命是不可能的。 由于设计和执行荧光寿命实验所需的先进专业知识的阻碍， 在荧光寿命指标和分子特异性之间的相关性，以及缺乏强大的模型， 从自发荧光特征确定代谢表型和线粒体功能。因此，博士。 沃尔什未来五年的目标将解决这些限制。目标1：确定灵敏度和 自体荧光寿命成像的特异性，以确定细胞代谢状态和途径利用。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 来报告细胞代谢。目标3：开发、测试和优化自体荧光 成像方案来量化线粒体动力学和功能。该提案的结果将使 具有高时间分辨率和特异性的代谢测量，用于评估活体细胞代谢 细胞此外，自体荧光成像将提供一个平台，以评估药物的影响， 药理学试剂，包括麻醉剂，对活细胞中的细胞代谢和线粒体功能的影响， 组织和体内。