Autofluorescence Across Scales: An Integrated Understanding Of Redox Cofactors As Intrinsic Probes Of Metabolic State

活细胞需要不断的能量输入来完成它们的任务。它通过一系列被称为新陈代谢的化学反应从食物的分子成分中释放出来。根据细胞的类型、环境和可用的燃料类型（如糖、脂肪或蛋白质），可以利用不同的代谢反应，即代谢途径。新陈代谢功能不正常是许多疾病的原因和结果。例如，人们早就知道，许多类型的癌症会代谢大量的糖，通过避开需要氧气的途径而低效地释放能量。这被称为Warburg效应。代谢功能障碍在糖尿病、心脏病和神经退行性疾病以及衰老的基本过程中也起着重要作用。我们对新陈代谢及其在疾病中的作用的了解主要来自于分解细胞并提取其内容物进行分析的实验。破坏细胞使研究组织的代谢如何随时间变化变得困难，并且代谢物可能在细胞环境外降解。此外，当复杂组织中存在一种以上的细胞类型时，这些方法对细胞特异性差异不敏感。为了继续推进我们对新陈代谢的理解，我们需要工具，使我们能够在一个完整的、活的组织的不同细胞类型内可视化代谢过程。我将利用代谢过程中关键分子的固有荧光来实现这一目标，也就是氧化还原辅因子。这些电子在不同的代谢反应之间传递电子，或者作为小的、可移动的载体，如烟酰胺腺嘌呤二核苷酸（NADH），或者作为蛋白质内的功能基团，如黄素。这些分子的荧光特性根据它们所结合的特定酶或它们是否携带电子而变化。这种自身荧光穿过组织的图像将被拍摄并分析其特性，以提取有关每个细胞代谢状态的信息。作为一名科学家，我的专业知识涵盖了使用激光来研究分子的动态行为，并将这些方法应用于研究活组织中的代谢过程，我很有能力研究使用自体荧光来研究代谢状态。我将进行实验并进行计算机模拟，以分析细胞尺度的代谢过程如何影响氧化还原辅助因子的分子尺度荧光。然后，我将找出最准确和用户友好的方法，从自身荧光测量中提取代谢信息，以建立一种新的实验方法，供更广泛的生物医学研究界使用。

Living cells require the constant input of energy to carry out their defined roles. This is released from the molecular constituents of food by a set of chemical reactions known as metabolism. Different sets of metabolic reactions, known as metabolic pathways, can be utilised depending on the type of cell, its environment and the type of fuel available, such as sugars, fats or proteins. The incorrect functioning of metabolism is known to be both a cause and effect of a wide range of diseases. For example, it has long been known that many types of cancer will metabolise vast amounts of sugar, releasing its energy inefficiently by avoiding pathways that require oxygen. This is known as the Warburg effect. Metabolic dysfunction also plays a major role in diabetes, heart disease and neurodegenerative diseases, as well as the fundamental processes of ageing.Our understanding of metabolism and its role in disease has largely resulted from experiments whereupon cells are broken down and the contents are extracted for analysis. Destroying the cell makes it difficult to investigate how the metabolism of a tissue changes with time, and the metabolites may degrade outside of the cellular environment. Furthermore, when more than one cell type is present in a complex tissue, these methods are insensitive to cell-specific differences. To continue advancing our understanding of metabolism, we require tools that allow us to visualise metabolic processes inside the different cell types of an intact, living tissue. I will achieve this by exploiting the intrinsic fluorescence of key molecules involved in metabolism, known as redox cofactors. These transfer electrons between different metabolic reactions, either as small, mobile carriers such as nicotinamide adenine dinucleotide (NADH), or as a functional group within a protein, such as flavin. The fluorescence characteristics of these molecules change depending on the specific enzyme they are bound to or whether they are carrying an electron. Images of this autofluorescence across a tissue will be taken and its properties analysed to extract information on the metabolic state of each cell.As a scientist whose expertise spans the use of lasers to study the dynamic behaviour of molecules and the application of these methods to investigate metabolic processes in living tissues, I am well equipped to investigate the use of autofluorescence for studying metabolic state. I will perform experiments and carry out computer simulations to analyse how cellular-scale metabolic processes impact the molecular-scale fluorescence of redox cofactors. I will then work out the most accurate and user-friendly ways to extract metabolic information from autofluorescence measurements to establish a novel experimental method for use by the wider biomedical research community.