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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

基本信息

批准号：
10663357
负责人：
Alexandra Walsh
金额：
$ 35.94万
依托单位：
TEXAS ENGINEERING EXPERIMENT STATION
依托单位国家：
美国
项目类别：
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/10663357
关键词：
Abnormal Cell 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 Protocols documentation Reporting Research Research Personnel Resolution Sampling Sensitivity and Specificity Signal Transduction Specificity Techniques Testing Tissues Toxic effect design experimental study fluorodeoxyglucose positron emission tomography in vivo insight metabolic abnormality assessment metabolic phenotype metabolomics mitochondrial dysfunction mitochondrial metabolism pharmacologic preclinical study prevent programs side effect temporal measurement tool whole body imaging

项目摘要

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。沃尔什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，许多药物和药剂，包括麻醉剂，通过改变代谢信号或因受损而产生的间接毒性具有直接作用机制线粒体功能。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。然而，这些现有工具的空间和时间分辨率以及分析的标签依赖性性质有限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。自发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寿命指标和分子特异性之间的相关性，以及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。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。目标 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量化线粒体动力学和功能的成像方案。该提案的成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，自发荧光成像将提供一个平台来评估药物和药物的影响药物，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。