Comparison of NADH FLIM and Intensity Imaging to Assess Cellular Energetics

描述（由申请人提供）：代谢稳态，或将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的产生。光学成像模式特别有吸引力，因为光是微创的，很容易传递到身体的各种组织，并且能够提供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烟酰胺腺嘌呤二核苷酸（NADH）的还原形式和直接参与线粒体能量产生的黄蛋白的固有荧光发射。这已被广泛应用于各种组织，并有助于我们目前对代谢状态的调节和维持的理解的发展。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，代谢成像采用荧光强度作为呼吸链电子供体浓度的替代物。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，并且可以恰当地表达为荧光团寿命和浓度的产物。由于自由种群与酶结合种群比例的变化也可能导致寿命的变化，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）可以提供更准确的细胞能量学测量。虽然FLIM是一种很有前途的新选择，但它还没有在控制良好的现实细胞环境中得到适当的评估。利用易于操作但相关的体外培养，我们建议系统地比较从NADH FLIM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点和局限性，我们将能够正确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。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概况。

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.