ASSESSMENT OF CELLULAR ENERGETICS BY NADH FLIM

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 代谢稳态，或密切匹配能量生产与需求的能力，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的寿命和浓度的乘积。由于寿命的变化也可能发生的结果，在自由酶结合的荧光基团的人口比例的变化，强度的变化是难以解释的。 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、诊断和开发针对各种人类疾病的治疗干预措施。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.