Comparison of NADH FLIM and Intensity Imaging to Assess Cellular Energetics

NADH FLIM 和强度成像评估细胞能量的比较

• 批准号: 7516180
• 负责人: MICHAEL G NICHOLS
• 金额: $ 21.68万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7516180
• 关键词: Age; Anoxia; Binding; Brain; Cell Respiration; Cell physiology; Cells; Cochlea; Complex; Cultured Cells; Development; Diagnosis; Disease; Electrodes; Environment; Enzymes; Experimental Designs; Feedback; Flavoproteins; Fluorescence; Fostering; Functional disorder; Future; Glucose; Hair Cells; Health; Homeostasis; Image; Imaging Techniques; In Vitro; Intervention; Invasive; Life; Light; Maintenance; Measurement; Measures; Metabolic; Metabolism; Methods; Mitochondria; Modality; Modeling; Monitor; NADH; Nicotinamide adenine dinucleotide; Noise-Induced Hearing Loss; Optics; Organ; Organ of Corti; Output; Oxygen; Oxygen Consumption; Population; Production; Range; Rate; Regulation; Resolution; Respiration; Respiratory Chain; Slice; Techniques; Technology; Testing; Thick; Time; Tissues; Work; base; body system; clinically relevant; electron donor; experience; fluorescence imaging; fluorophore; hearing impairment; human disease; monolayer; novel; optical imaging; research study; simulation; tissue/cell culture; trafficking; tumor; two-photon

DESCRIPTION (provided by applicant): Metabolic homeostasis, or the ability to closely match energy production with demand, is a fundamental requirement for the health and normal functioning of the cells, tissues, and organs of the body. Therefore, several technologies have been developed to assess cellular energy production. Optical imaging modalities are particularly attractive, because light is minimally invasive, easily delivered to the various tissues of the body, and capable of providing rapid feedback with high spatial resolution. One approach to characterizing the metabolic state has been to use the intrinsic fluorescence emission from the reduced form of nicotinamide adenine dinucleotide (NADH) and flavoproteins that directly participate in mitochondrial energy production. This has been extensively applied to a variety of tissues and has been instrumental in the development of our current understanding of the regulation and maintenance of the metabolic state. However, interpreting the observed changes in tissue fluorescence can be problematic, often requiring assumptions or the need of additional measurements for effective application. Traditionally, metabolic imaging has employed fluorescence intensity as a surrogate for the concentration of electron donors to the respiratory chain. Recent studies, however, have shown that these measurements of fluorophore concentration may be error prone. Fundamentally, this is because the fluorescence intensity is dependent on the local environment of the fluorophore and is properly expressed as a product of both the fluorophores lifetime and concentration. Since lifetime changes can also occur as a result of changes in the ratio of the free to enzyme-bound populations, changes in intensity are difficult to interpret. Other studies have suggested that NADH fluorescence lifetime imaging (FLIM) may provide a more accurate measurement of cellular energetics. While FLIM is a promising, novel alternative, it has yet to be properly evaluated in well controlled, yet realistic cellular environments. Using easily manipulated yet relevant in vitro cultures, we propose to systematically compare measurements of the cellular metabolic state obtained from NADH FLIM with the traditional assessment made using fluorescence intensity alone. By establishing the advantages and limitations of this new technique, we will be able to properly deploy metabolic imaging techniques to better characterize, diagnose and develop treatment interventions for a broad range of human diseases. The health and normal functioning of living cells and tissues requires that energy output be closely regulated to energy demand. Minimally invasive imaging techniques that monitor cellular energetics can help to explain, diagnose, and develop treatments for disease. This project will determine whether a novel fluorescence lifetime-based imaging strategy can provide a more accurate profile of cellular metabolism than traditional techniques that rely on fluorescence intensity alone.

描述（由申请人提供）：代谢稳态，或使能量产生与需求紧密匹配的能力，是身体细胞、组织和器官健康和正常功能的基本要求。因此，已经开发了几种技术来评估细胞能量产生。光学成像模式特别有吸引力，因为光是微创的，容易递送到身体的各种组织，并且能够提供具有高空间分辨率的快速反馈。表征代谢状态的一种方法是使用来自还原形式的烟酰胺腺嘌呤二核苷酸（NADH）和直接参与线粒体能量产生的黄素蛋白的固有荧光发射。这已被广泛应用于各种组织，并已在我们目前的理解的发展和代谢状态的调节和维持的工具。然而，解释观察到的组织荧光变化可能是有问题的，通常需要假设或需要额外的测量来有效应用。传统上，代谢成像采用荧光强度作为呼吸链电子供体浓度的替代。然而，最近的研究表明，这些荧光团浓度的测量可能容易出错。从根本上说，这是因为荧光强度取决于荧光团的局部环境，并适当地表示为荧光团寿命和浓度的乘积。由于游离与酶结合群体比例的变化也可能导致寿命变化，因此难以解释强度变化。其他研究表明，NADH荧光寿命成像（FLIM）可以提供更准确的测量细胞能量。虽然FLIM是一个有前途的，新颖的替代品，它还没有得到适当的评估，在良好的控制，但现实的细胞环境。使用易于操作，但相关的体外培养，我们建议系统地比较测量的细胞代谢状态从NADH FLIM与传统的评估单独使用荧光强度。通过确定这项新技术的优势和局限性，我们将能够正确部署代谢成像技术，以更好地表征、诊断和开发各种人类疾病的治疗干预措施。活细胞和组织的健康和正常功能要求能量输出与能量需求密切相关。监测细胞能量学的微创成像技术可以帮助解释，诊断和开发疾病的治疗方法。该项目将确定一种新的基于荧光寿命的成像策略是否可以提供比仅依赖荧光强度的传统技术更准确的细胞代谢谱。