In situ quantification of metabolic function using fluorescence lifetime imaging

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.

Correct metabolic function is pivotal to cell health, while defects in metabolism are implicated in a wide range of major diseases. In order to understand the metabolism of specific tissues, the availability of reliable, unambiguous and accurate tools that report metabolic state in live samples is essential. Over the past 20 years, this role has been played by fluorescence imaging, providing important information about the metabolic state of living cells. However, the standard measurements of fluorescence intensity in current use yield, at best, only semi-quantitative measurements. We therefore propose to develop novel approaches, employing fluorescence lifetime imaging microscopy (FLIM) to make accurate quantifications of three variables fundamental to cell metabolism, extending the range of approaches available to study the metabolic state of live biological models. FLIM measures the fluorescence lifetime of a target fluorophore at each pixel of an image. As the lifetime of a probe is highly sensitive to its immediate environment, FLIM is potentially a rich source of biochemical data. However, to clarify and optimise the information content of these measurements, it is necessary to understand the relationship between probe photophysics and the underlying biochemistry and physiology. Our proposal therefore combines two research laboratories with expertise in molecular physics and ultrafast spectroscopy, and in metabolism and fluorescence imaging of live samples, in order to (A) develop FLIM of NAD(P)H as a label-free technique for monitoring redox state in complex tissues, (B) establish novel protocols for the absolute measurement of mitochondrial membrane potentials inside live cells and (C) accurately quantify ATP concentrations in subcompartments of intact tissue using genetically-encoded FRET probes. Such techniques will find widespread use in quantifying metabolic function in situ, placing FLIM at the forefront of investigations into health and disease.

Metabolism plays a major role in health and disease. This project aims to provide biomedical researchers with new tools to study metabolic properties of fundamental importance - NAD(P)H levels, the mitochondrial membrane potential and ATP production and consumption - by the application of fluorescence lifetime imaging microscopy (FLIM). For this reason, the primary beneficiary of this proposal is clearly the wider academic community, and these links are outlined in the Academic Beneficiaries section. However, there are a number of other non-academic areas in which this proposal will provide impact.
Firstly, a number of systems are in development for high-throughput screening of live cells using fluorescence lifetime measurements. Our protocols will be scaleable to these systems, allowing their use in the pharmaceutical industry for large-scale assays of treatments developed to target metabolism. As altered metabolic states in pathophysiology are so ubiquitous, the application of these techniques for drug discovery would have a clear impact on economic wealth creation. This is particularly true of our approach to quantify ATP concentrations using FRET. The acceptor rise amplitude technique that we will apply in our protocols will allow determination of the concentration of a target molecule with unprecedented accuracy. Similar fluorescent protein based FRET probes are now routinely developed for a given target of interest. As such, proving the principle of acceptor rise amplitude determination in the high-throughput measurement of ATP concentrations will open the door for large-scale yet accurate determination of concentrations of a desired molecular species in live biological samples.
Secondly, as our proposal crucially depends on the collaboration between biological and physical science laboratories, it represents a clear demonstration of the increasing need for interdisciplinarity in modern scientific research. Thus, given proper engagement, the public understanding of science will be increased by using our research as a prime example of the context in which 21st century biology is beginning to take place. In particular, our work will demonstrate to students that the borders between disciplines introduced at school cease to exist at the cutting edge, so those considering a career in science need not "give up" their interest in two of the three core subjects to pursue the other. The early exposure to collaboration between disciplines will inform the paths of these students through higher education. This will increase the likelihood that the next generation of scientists will be produced with a wide knowledge and skills range, having a positive impact on science in the United Kingdom and thus the economic prosperity of the nation. Such impact will also be felt locally and in the shorter term, with the junior postdoctoral researcher involved in this project gaining experience in a diverse array of experimental approaches in both the life and physical sciences, priming him for a career at the interface between disciplines.
Finally, there is obvious potential for the techniques developed in this proposal to increase wellbeing. The new tools proposed in this work will open up avenues for the fundamental understanding of metabolism in health and disease. In particular, their ability to quantitatively probe the function of mitochondria in situ will enhance the understanding of ageing in specific tissues, as these organelles are responsible for the majority of free radical species produced in the cell. With the nation's rapidly ageing population set to be a significant factor to social welfare in the coming decades, an increased understanding of the biology of ageing and neurodegenerative disease, facilitated by our techniques, will have a huge impact on the quality of life of the nation.
The means by which we will facilitate impact to potential beneficiaries are discussed in the attached Pathways to Impact document.