In situ quantification of metabolic function using fluorescence lifetime imaging

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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.