In situ quantification of metabolic function using fluorescence lifetime imaging

代谢是细胞内发生的一系列化学转化，使生物体保持活力，例如打破营养分子中原子之间的键以提供能量，或从简单的单位构建复杂的分子以供生长。这些过程是生命的基础，它们的失败与癌症，糖尿病和神经退行性疾病等疾病有关。为了理解代谢在特定过程中的作用，我们需要提供活组织中代谢特性的定量测量的工具。然而，目前的技术是有限的，在我们的知识中留下了空白。我们建议开发新的工具，将提供准确和可靠的定量代谢特性，使用荧光寿命成像（FLIM）的技术。在FLIM中，激光扫描整个活组织激发荧光分子添加到样品中或自然存在。激光束是每秒高达8000万个光脉冲的序列。当光粒子（光子）被荧光分子吸收时，FLIM测量光重新发射所需的时间。发生这种情况所需的平均时间，即荧光寿命，对分子的直接环境非常敏感。原则上，通过选择合适的荧光探针并测量这些延迟时间，可以在生命系统中研究代谢反应。汇集了研究小组在快激光光谱学、生物物理学、代谢和活细胞荧光成像方面的专业知识，本提案的目标是创建使用FLIM测量三个基本代谢特性的方法。首先，我们将研究两种天然存在于所有组织中的荧光分子：NADH和NADPH。这些分子的荧光寿命随着疾病的进展而变化，并且已经基于这种观察设计了用于诊断的仪器。然而，由于缺乏对导致这种变异的生化变化的了解，它们的临床应用受到限制。我们将通过激活和抑制活细胞中的特定途径并观察这些分子的寿命如何变化来确定哪些代谢途径决定NADH和NADPH的荧光寿命。这将为FLIM应用于疾病诊断奠定基础，通过避免需要侵入性活检而使患者受益。第二，我们提出了一种方法来测量由“细胞发电站”，线粒体产生的电压，使用FLIM观察荧光染料（TMRM）被细胞吸收时的行为。线粒体负责释放储存在碳水化合物、脂肪和蛋白质中的能量。类似于电池，线粒体膜上的电压控制着这一过程，但它也决定了细胞死亡和自由基的产生，这与衰老有关。膜电位是线粒体功能的核心，在活体组织中对其进行精确测量将是理解其在疾病中作用的关键。最后，我们将开发一种使用FLIM测量ATP水平的方法，ATP被视为生命的“通用能量货币”。ATP由线粒体产生，并为大多数细胞过程提供动力，从DNA产生到细胞运动。为了测量活组织中的这个量，我们将引入编码两个荧光蛋白的DNA，它们连接到ATP结合位点。当ATP附着时，荧光蛋白被带到附近，允许能量在它们之间转移。FLIM可以用来测量有多少蛋白质正在经历这个过程，称为福斯特共振能量转移（FRET）。然后可以从该分数计算ATP浓度。由于ATP是生命化学的核心，因此创建一种方法来准确测量其在活体样本中的生产和消耗，将为找到治疗世界上一些最严重疾病的最终目标做出重大贡献。

Metabolism is the set of chemical transformations taking place inside a cell that keep an organism alive, such as breaking of bonds between atoms in nutrient molecules to provide energy or building complex molecules from simpler units for growth. These processes are fundamental to life, and their failure is associated with diseases such as cancer, diabetes and neurodegeneration. To understand the role of metabolism in particular processes, we require tools that provide quantitative measures of metabolic properties in living tissues. However, current techniques are limited, leaving gaps in our knowledge. We propose to develop new tools that will provide accurate and reliable quantification of metabolic properties, using the technique of fluorescence lifetime imaging (FLIM).In FLIM, a laser is scanned across a living tissue to excite fluorescent molecules added into the sample or naturally present. The laser beam is a train of up to 80 million light pulses every second. When a particle of light (a photon) is absorbed by a fluorescent molecule, FLIM measures the time taken for the light to be re-emitted. The average time taken for this to occur, the fluorescence lifetime, is extremely sensitive to the immediate environment of the molecule. The reactions of metabolism can, in principle, be studied in living systems by choosing appropriate fluorescent probes and measuring these delay times.Pooling the expertise of research groups working in fast laser spectroscopy, photophysics, metabolism and fluorescence imaging of live cells, the objective of this proposal is to create methods to measure three fundamental metabolic properties using FLIM.First, we will study two fluorescent molecules that are naturally present in all tissues; NADH and NADPH. The fluorescence lifetime of these molecules changes as disease progresses, and instruments have been designed for diagnosis based on this observation. However, their clinical use has been limited by a lack of understanding of the biochemical changes that cause this variation. We will determine which metabolic pathways dictate the fluorescence lifetime of NADH and NADPH by activating and inhibiting particular pathways in live cells and observing how the lifetimes of these molecules change. This will lay foundations for the application of FLIM to diagnose disease, benefitting patients by obviating the need for invasive biopsy.Second, we propose a method to measure the voltage produced by the "cell powerhouses", the mitochondria, using FLIM to observe the behaviour of a fluorescent dye (TMRM) when it is taken up by cells. Mitochondria are responsible for releasing the energy stored in carbohydrates, fats and proteins. Akin to a battery, the voltage across the mitochondrial membrane governs this process, but it also determines cell death and the production of free radicals, implicated in ageing. The membrane potential is central to mitochondrial function and the ability to make accurate measurements of it in live tissues will be key to understanding its role in disease.Finally, we will develop a method for measuring levels of ATP, regarded as life's "universal energy currency", using FLIM. ATP is produced by the mitochondria and powers the majority of cellular processes, from DNA production to cell motion. To measure this quantity in live tissues, we will introduce DNA coding for two fluorescent proteins attached to an ATP binding site. The fluorescent proteins are brought into proximity when ATP attaches, allowing energy to be transferred between them. FLIM can be used to measure how many proteins are undergoing this process, known as Förster resonance energy transfer (FRET). The ATP concentration can then be calculated from this fraction. As ATP is so central to life's chemistry, creating a method to accurately measure its production and consumption inside living samples will make a major contribution to the ultimate aim of finding a cure for some of the world's most severe diseases.