Datta Rupsa, Alfonso-García Alba, Cinco Rachel, Gratton Enrico
Laboratory of Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine.
Department of Biomedical Engineering, University of California, Irvine.
Sci Rep. 2015 May 20;5:9848. doi: 10.1038/srep09848.
Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. Further, we performed spontaneous Raman spectral analysis at single points of the sample which provided molecular vibration information characteristics of lipid droplets.
活性氧（ROS）的存在超过正常生理水平会导致氧化应激。这可能会引发一系列病理状况，包括炎症、糖尿病、癌症、心血管疾病和神经退行性疾病。氧化应激的生物标志物在理解这些疾病的发病机制和治疗方面发挥着重要作用。存在多种荧光生物标志物。然而，一种非侵入性且无标记的识别技术对于体内测量将是有利的。在这项工作中，我们建立了一种通过荧光寿命成像（FLIM）来识别细胞和组织中氧化应激的光谱方法。我们鉴定出一种具有特征性荧光寿命分布的自发荧光内源性物质作为氧化应激的探针。为了证实我们的假设，即这些物质是ROS引发的脂质氧化产物，我们通过将FLIM与THG和CARS显微镜相结合，将来自脂滴的光谱信号关联起来，THG和CARS显微镜是用于选择性脂质体成像的成熟技术。此外，我们在样品的单点进行了自发拉曼光谱分析，该分析提供了脂滴的分子振动信息特征。
