Autofluorescence lifetime metrology for label-free readouts of heart disease and arthritis

This project aims to provide new label-free detection and imaging tools for minimally invasive diagnosis of arthritis and heart disease. When biological tissue is illuminated with light at appropriate wavelengths, certain naturally occurring biomolecules can absorb this excitation energy and emit new radiation called fluorescence. By analysing such autofluorescence signals, it is possible to detect particular biochemical or structural changes in tissue, which may be exploited to detect the early onset of diseases such as arthritis, heart disease and cancer, which can cause changes in the concentration, distribution and interaction of these autofluorescent biomolecules. Unfortunately, biological tissue is heterogeneous, typically containing many kinds of biomolecule in unknown quantities that can interact with autofluorescence measurements. It also strongly scatters optical radiation, making quantitative fluorescence measurements unreliable. It is therefore desirable to analyse tissue autofluorescence in a way that avoids artefacts arising from unknown variations in molecular concentrations and light intensity. One way to do this is to exploit the fact that excited molecules can radiate fluorescence at different rates, depending on the particular biomolecule or on how it is interacting with its surroundings. By observing the fluorescence decay times (lifetimes), it is possible to distinguish different chemical species or different molecular environments that can correlate with different tissue structures. Although this technique is attracting increasing interest in laboratory-based research, there is very little work, to date, on translating this to clinical practice. Here, we propose to develop fibre-optic-based probes that clinicians can use to measure fluorescence lifetimes in situ in biological tissue samples or live subjects including patients. We will also develop a fluorescence lifetime imaging (FLIM) system that will be able to rapidly map the spatial variation of autofluorescence lifetime, which can provide diagnostic functional images for medical research and clinical practice.We will develop one fibre-optic autofluorescence lifetime (AFL) probe to be applied to changes in tissue matrix components associated with arthritis, which leads to loss of joint function and pain in ~9 million people in the UK's aging population. Arthritis is caused by the degradation of cartilage in our limb joints. Currently, there is no way to examine the integrity of cartilage tissue without invasive surgical biopsy. In preliminary work, we have shown that degraded cartilage exhibits significantly different AFL compared to healthy cartilage. Here we aim to investigate how AFL measurements can provide label free information on the structure and health of cartilage. We would also investigate the prognostic value of FLIM arthroscopy of cartilage in early rheumatoid and osteoarthritis and monitor the effect of drugs applied to repair cartilage.A second AFL probe will be applied to the study of heart disease - the major cause of death in the developed world - which is characterised by abnormalities of heart muscle energetics. Energetic inefficiency accelerates further disease progression, manifesting as changes in the mechanical and electrical properties of the heart. Two important autofluorescent molecules, NAD(P)H and flavins, are intimately involved in metabolism but, apart from our preliminary results presented below, no AFL measurements have been made in situ in live heart tissue. We aim to investigate how cardiac AFL can provide a quantitative label-free readout of the metabolic state of the beating heart and to identify signatures of disease through AFL measurements and FLIM of cardiac tissue matrix adaptations associated with heart disease and electrical disturbances. These label-free readouts of diseased heart tissue could provide a novel means to determine treatment of patients following a heart attack.

This project aims to provide new non-invasive tools for improved patient prognosis and treatment, particularly for arthritis and heart disease, and so addresses the needs of the aging UK population. The autofluorescence lifetime (AFL) technologies to be developed and validated would find other biomedical applications, e.g. for research, diagnosis and monitoring of therapies in cancer, inflammation, wound healing and any condition where label-free readouts of tissue matrix properties or metabolism are useful. They could also be used with fluorescence labels, including in genetically manipulated live disease models to provide readouts of signal pathways for drug discovery. Other bio-applications include tissue engineering, stem cell culture and bio-reactors. Some of these are being addressed by lab-based FLIM microscopy but our fit-for-purpose technology should lower the cost and increase the potential for real-world deployment. AFL technologies are applicable in the pharmaceutical sector, e.g. to test drugs in patients and animals - permitting longitudinal studies and therefore reducing the required numbers of animals. For agriculture, AFL may be useful to study plant disease, to monitor the effects of chemical intervention and to assess crops before harvest. For food safety, AFL may help detect dangerous bacteria or decomposition. Beyond the life sciences, our proposed instrumentation could be used to rapidly assess, in situ, devices such as OLED's and photovoltaics, including during manufacturing for quality control. The most direct impact of this project is envisaged to be medical research addressing the label-free detection, diagnosis and monitoring of arthritis and heart disease, although we anticipate patient-orientated follow-on studies and clinical trials to deliver new clinical tools. We also note the valuable training of project research staff. For both arthritis and heart disease, there are unmet needs for minimally invasive functional and spatially-resolved readouts of biological tissue to improve patient prognosis and more effectively target treatment. For arthritis, the proposed AFL instrumentation could enable the early detection of disease - before significant irreversible cartilage degradation. This would help the NHS budget by reducing the number of joint replacements and significantly improve patient quality of life. The ability to monitor cartilage integrity in situ also enables monitoring of potential therapies such as inhibitors that block the action of cartilage-degrading enzymes that otherwise can only be studied using invasive biopsy and/or the sacrifice of animals at each endpoint. Currently there are no such therapies targeting cartilage degradation available for arthritis. For heart disease the use of AFL to read out changes in metabolism and tissue matrix properties could address the current inability to accurately predict which heart disease patients are at risk of both sudden cardiac death from malignant arrhythmias and progressive mechanical deterioration and death from 'pump failure'. Better patient selection and targeting of expensive treatments, such as implantable defibrillators, arrhythmia ablation and the emerging stem cell and gene therapies, would significantly improve patient outcomes and reduce costs to the NHS. Because this AFL instrumentation represents a relatively cost-effective add-on to current clinical diagnostic and therapeutic approaches, it presents new commercial opportunities, e.g. for medical device manufacturers. Kentech Instruments Ltd, in particular, are keen to develop products for clinical AFL imaging based on their gated intensifier technology and will work with us to establish the requirements for clinical devices. For the single-point AFL fibre-optic probe, we are seeking commercial exploitation partners and want to use the data from this project to validate the clinical applications and strengthen our case for commercial investment.