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; 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.

项目摘要/摘要 这是提交早期提交的最大化研究者研究奖（MIRA）的申请 研究人员（ESI），Alex Walsh博士。沃尔什博士的研究计划着重于表征和 开发自荧光寿命显微镜，用于定量细胞代谢和线粒体 活细胞的功能。异常细胞代谢和线粒体功能障碍是许多疾病的标志 和病理状态。此外，包括麻醉在内的许多药物和药物 通过改变的新陈代谢信号传导或间接毒性，具有直接的作用机理 线粒体功能。当前评估细胞代谢的研究测定法包括基于细胞的测定法 基因组，代谢组和氧气消费测定和全身成像测定法 荧光脱氧葡萄糖 - 固醇发射断层扫描和碳13磁共振成像。然而， 这些现有工具具有有限的空间和临时分辨率以及分析的标签性质 防止对同一样品的动态代谢状态和多次纵向评估的评估。 线粒体内烟酰胺腺苷（NADH）减少的自动荧光寿命成像 线粒体中的细胞质和黄素腺嘌呤二核苷酸（FAD）呈现出独特的，无标签的，高 - 评估细胞代谢的分辨率技术。自荧光寿命显微镜不取决于 化学或抗体标签是非接触的，广泛适用于任何细胞或组织，非常适合纵向 研究，与次要测定兼容。但是，当前采用自动荧光寿命 受到设计和执行荧光寿命实验所需的高级专业知识的阻碍 在荧光寿命指标与分子特异性之间的相关性中，缺乏可靠的模型 通过自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 自荧光寿命成像的特异性，以识别细胞代谢状态和途径利用率。目标 2：量化常见实验室模型的自动荧光寿命成像特征以确定鲁棒性 自动荧光指标报告细胞代谢。目标3：开发，测试和优化自动荧光 量化线粒体动力学和功能的成像协议。该提案的结果将启用 具有高临时分辨率和特异性的代谢测量，以评估生命中的细胞代谢 细胞。此外，自荧光成像将提供一个平台来评估药物的影响和 药理学剂，包括麻醉药，有关活细胞中细胞代谢和线粒体功能的药理剂， 组织和体内。