ASSESSMENT OF CELLULAR ENERGETICS BY NADH FLIM

NADH FLIM 对细胞能量的评估

• 批准号: 8360017
• 负责人: MICHAEL G NICHOLS
• 金额: $ 1.43万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360017
• 关键词: Binding; Cell physiology; Development; Diagnosis; Environment; Enzymes; Feedback; Flavoproteins; Fluorescence; Funding; Grant; Health; Homeostasis; Image; Imaging Techniques; In Vitro; Intervention; Light; Maintenance; Measurement; Metabolic; Mitochondria; NADH; National Center for Research Resources; Nebraska; Nicotinamide adenine dinucleotide; Organ; Population; Principal Investigator; Production; Regulation; Research; Research Infrastructure; Resolution; Resources; Respiratory Chain; Source; Techniques; Technology; Tissues; United States National Institutes of Health; cost; electron donor; fluorophore; functional genomics; human disease; imaging modality; minimally invasive; novel; optical imaging

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 fluorophore's lifetime and concentration. Since lifetime changes can also occur as a result of changes in the ratio of the free to enzyme-bound fluorophores 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 disease.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 代谢动态平衡，即能量生产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运作的基本要求。因此，已经开发了几种技术来评估细胞的能量产生。光学成像方式特别有吸引力，因为光是微创的，很容易传递到身体的各种组织，并且能够以高空间分辨率提供快速反馈。一种表征代谢状态的方法是使用直接参与线粒体能量产生的还原形式的烟酰胺腺嘌呤二核苷酸(NADH)和黄素蛋白的本征荧光发射。这已被广泛应用于各种组织，并有助于发展我们目前对代谢状态调节和维持的理解。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才能有效应用。传统上，代谢成像使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，并且可以恰当地表示为荧光团寿命和浓度的乘积。由于寿命的变化也可能发生在游离态与酶结合态荧光团的比例发生变化时，因此强度的变化很难解释。其他研究表明，NADH荧光寿命成像(FLIM)可能提供更准确的细胞能量学测量。虽然Flim是一种有希望的、新颖的替代方案，但它还没有在控制良好的、但现实的蜂窝环境中进行适当的评估。使用易于操作但相关的体外培养，我们建议系统地比较从NADH薄膜获得的细胞代谢状态的测量结果与仅使用荧光强度进行的传统评估。通过确定这项新技术的优势和局限性，我们将能够适当地部署代谢成像技术，以更好地表征、诊断和开发针对广泛人类疾病的治疗干预措施。