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

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.

Metabolism encompasses the complex network of chemical reactions that sustain life at the cellular level. Dysfunction of these pathways is known to play a major role in disease. Altered metabolism is also widely acknowledged to be a significant part of the fundamental process of ageing. The classic biochemical approaches upon which knowledge of metabolism has been successfully developed are generally insensitive to the spatial organisation of cells and tissues. To understand metabolic processes in biological models that more truly represent whole living systems, researchers require new experimental tools. Fluorescence imaging approaches would be ideal for this purpose but introducing extrinsic probes into complex tissue preparations can be challenging. I will therefore investigate how cellular autofluorescence, resulting from the mobile NADH and NADPH redox carriers and flavin prosthetic groups in flavoproteins, can report the metabolic state of a tissue.

This proposal investigates how time- and polarisation-resolved NAD(P)H and flavin fluorescence relates to the metabolic pathways active in a tissue. To do this, the interrelationships of biochemical processes acting at scales between these molecular and cellular phenomena must be understood. I will achieve this by combining my existing skills in fluorescence spectroscopy and imaging of living tissues with the development of new skills in molecular dynamics and quantum mechanics/molecular mechanics. I will investigate how the dynamic binding equilibrium of cofactors and reactants affects the fluorescence lifetime of NAD(P)H, how quenching of flavin fluorescence differs between different flavoproteins, and how the time-resolved anisotropy of autofluorescence changes with metabolic state. Ultimately, I will establish protocols for the analysis of time- and polarisation-resolved autofluorescence imaging that maximise the metabolic information available to end-users from the biomedical research community.