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

本项目旨在为关节炎和心脏病的微创诊断提供新的无标签检测和成像工具。当生物组织被适当波长的光照射时，某些天然存在的生物分子可以吸收这种激发能量并发出称为荧光的新辐射。通过分析这些自身荧光信号，可以检测组织中特定的生化或结构变化，这可能被用于检测诸如关节炎、心脏病和癌症等疾病的早期发作，这些疾病可能导致这些自身荧光生物分子的浓度、分布和相互作用的变化。不幸的是，生物组织是不均匀的，通常含有多种未知数量的生物分子，可以与自身荧光测量相互作用。它还会强烈散射光辐射，使荧光定量测量不可靠。因此，需要以一种避免由分子浓度和光强度的未知变化引起的伪影的方式分析组织自身荧光。一种方法是利用受激分子以不同速率辐射荧光这一事实，这取决于特定的生物分子或它与周围环境的相互作用方式。通过观察荧光衰减时间（寿命），可以区分与不同组织结构相关的不同化学物质或不同分子环境。尽管这项技术在实验室基础研究中吸引了越来越多的兴趣，但迄今为止，将其转化为临床实践的工作很少。在这里，我们建议开发基于光纤的探针，临床医生可以使用它来原位测量生物组织样本或活体受试者（包括患者）的荧光寿命。我们还将开发一种荧光寿命成像（FLIM）系统，该系统将能够快速绘制自身荧光寿命的空间变化，为医学研究和临床实践提供诊断功能图像。我们将开发一种光纤自身荧光寿命（AFL）探针，应用于与关节炎相关的组织基质成分的变化，关节炎导致英国约900万人的关节功能丧失和疼痛。关节炎是由我们肢体关节的软骨退化引起的。目前，没有办法检查软骨组织的完整性没有侵入性手术活检。在初步工作中，我们已经表明，与健康软骨相比，降解软骨表现出明显不同的AFL。在这里，我们的目的是研究AFL测量如何提供软骨结构和健康的无标签信息。我们还将探讨FLIM关节镜检查软骨在早期类风湿和骨关节炎中的预后价值，并监测软骨修复药物的效果。第二个AFL探针将应用于心脏病的研究——在发达国家，心脏病是导致死亡的主要原因——其特征是心肌能量代谢异常。能量效率低下加速了疾病的进一步发展，表现为心脏机械和电特性的变化。两种重要的自荧光分子，NAD(P)H和黄素，与代谢密切相关，但除了我们下面提出的初步结果外，没有在活体心脏组织中原位进行AFL测量。我们的目标是研究心脏AFL如何提供跳动心脏代谢状态的定量无标记读数，并通过AFL测量和与心脏病和电干扰相关的心脏组织基质适应的FLIM来识别疾病的特征。这些患病心脏组织的无标签读数可以提供一种新的方法来确定心脏病发作后患者的治疗方法。

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.