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.

项目概要/摘要 这是早期阶段提交的最大化研究者研究奖（MIRA）申请 研究员 (ESI) Alex Walsh 博士的研究项目侧重于表征和 开发用于定量细胞代谢和线粒体的自发荧光寿命显微镜 细胞代谢异常和线粒体功能障碍是许多疾病的标志。 此外，许多药物和药剂，包括麻醉。 通过代谢信号传导或因受损而产生的间接毒性具有直接作用机制 当前评估细胞代谢的研究分析包括基于细胞的分析，例如 基因组、代谢组学和耗氧量测定以及全身成像测定，例如 氟脱氧葡萄糖正电子发射断层扫描和碳 13 磁共振成像。 这些现有工具的空间和时间分辨率有限，并且检测具有标签依赖性 防止对动态代谢状态进行评估以及对同一样本进行多次纵向评估。 线粒体内还原型烟酰胺腺嘌呤二核苷酸 (NADH) 的自发荧光寿命成像 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 评估细胞代谢的分辨率技术不依赖于自发荧光寿命显微镜。 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，并与二次测定兼容然而，目前采用自发荧光寿命。 设计和执行荧光寿命实验所需的先进专业知识、默默无闻阻碍了 荧光指标和分子寿命特异性之间的相关性，并且缺乏稳健的模型 从自发荧光特征确定代谢表型和线粒体功能。 沃尔什未来五年的目标将解决这些限制 目标 1：确定敏感性和有效性。 自发荧光寿命成像的特异性，用于识别细胞代谢状态和途径利用。 2：量化常见实验室模型中的自发荧光寿命成像特征，以确定稳健性 报告细胞代谢的自发荧光指标 目标 3：开发、测试和优化自发荧光。 该提案的结果将使量化线粒体动力学和功能的成像方案成为可能。 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 此外，自发荧光成像将提供一个评估药物和细胞影响的平台。 药物，包括麻醉剂，影响活细胞中的细胞代谢和线粒体功能， 组织和体内。