TIME-RESOLVED AUTOFLUORESCENCE SPECTROSCOPY AS LABEL-FREE METHOD TO CHARACTERISE ACUTE CHANGES IN EX VIVO MODELS OF CARDIAC DISEASE
TIME-RESOLVED AUTOFLUORESCENCE SPECTROSCOPY AS LABEL-FREE METHOD TO CHARACTERISE ACUTE CHANGES IN EX VIVO MODELS OF CARDIAC DISEASE
复制标题
时间分辨自发荧光光谱作为无标记方法来表征心脏病离体模型的急性变化
DOI：
10.1136/heartjnl-2014-306916.38
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发表时间：
2014
期刊：
影响因子：
5.7
通讯作者：
Dyer B
中科院分区：
文献类型：
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作者：
Dyer B
时间分辨自发荧光光谱可以表征生物组织，并且已经在许多疾病应用中显示出潜力。它利用许多内源分子的光物理特性，例如NADH 和黄素蛋白，提供了辨别心肌能量状态的机会，而不会出现与引入外源化合物相关的毒性或后果问题。此外，来自细胞外基质分子的自发荧光，例如胶原蛋白，可以提供有关心脏结构变化的信息。我们报告了一种基于定制光纤探针的时间分辨分光荧光计的开发和应用，该仪器结合了时间相关的单光子计数检测和白光反射计，以表征与心脏健康和病理状态的组织学、形态、代谢和功能变化相关的自发荧光“指纹”。荧光寿命的测量本质上是比例测量，并且不受分子浓度和激发强度以及其他实验变量的影响。此外，荧光寿命还受到微环境变化的影响，例如：蛋白质结合状态。使用离体 Langendorff 系统灌注的雄性 SD 大鼠的移植心脏显示出稳定的荧光寿命。荧光寿命信号成功地从荧光衰减中获得，采集时间低至 1 秒。使用灌注液柱切换的离体 Langendorff 制剂的自发荧光信号的变化针对缺血再灌注模型以及葡萄糖和甘露醇之间的底物变化进行了表征。该技术有潜力成为一种无标记方法，用于观察与心脏疾病机制相关的生化变化，在心脏研究和临床诊断方面具有明显的潜在应用前景。
Time-resolved autofluorescence spectroscopy permits characterization of biological tissues and has already shown the potential in a number of disease applications. It harnesses the photo-physical properties of a number of endogenous molecules, e.g. NADH and flavoproteins, to offer an opportunity to discern the energetic state of the myocardium without the issues of toxicity or consequences associated with the introduction of exogenous compounds. Additionally, autofluorescence from extra-cellular matrix molecules, e.g. collagen, can provide information on structural changes to the heart. We report the development and application of a custom fibre-optic probe-based time-resolved spectrofluorometer combining time-correlated single photon counting detection and white-light reflectometry to characterize the autofluorescence ‘fingerprint’ associated with the changes in histology, morphology, metabolism and function in heart health and pathological states. Measurement of fluorescence lifetime is inherently ratiometric and unaffected by molecule concentration and excitation intensity amongst other experimental variables. Furthermore fluorescence lifetime is affected by changes in the microenvironment e.g. protein binding states. Explanted hearts from male SD rats perfused using the ex vivo Langendorff system demonstrated stable fluorescence lifetimes. Fluorescence lifetime signals were successfully derived from fluorescence decays with acquisition times as low as 1 second. The changes in the autofluorescence signal of ex vivo Langendorff preparations using perfusate column switching were characterised for an ischaemia-reperfusion model and for a change of substrate between glucose and mannitol. This technology has the potential to be a label-free method to observe biochemical changes associated with disease mechanisms in the heart, with clear potential application to cardiac research and clinical diagnostics.