Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, 106, Taiwan.
Anal Bioanal Chem. 2013 Oct;405(26):8549-59. doi: 10.1007/s00216-013-7251-0. Epub 2013 Aug 11.
The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R(2) > 0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K(i). Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.
脂质在巨噬细胞中的积累是促进动脉粥样硬化病变形成的一个关键因素。已经有几种方法，如生化分析和中性脂质染色，被用于检测细胞中的脂质。然而，由于缺乏适合的非侵入性化学检测工具，一种用于实时定量评估活巨噬细胞中脂质含量的方法，特别是其与药物的动力学过程，尚未被展示。在这里，我们通过相干反斯托克斯拉曼散射（CARS）显微镜展示了对活巨噬细胞中脂质滴（LDs）的无标记实时监测。此外，我们还建立了一种基于最大熵阈值（MET）的自动图像分析方法，用于定量细胞脂质含量。CARS 图像分析的结果与生化分析的测量结果有很好的相关性（R(2) > 0.9）。利用这种方法，我们监测了巨噬细胞中脂质积累和水解的过程。我们进一步研究了脂解抑制剂（二乙基伞形酮磷酸酯，DEUP）的作用，并确定了抑制常数 K(i)等动力学参数。我们的工作表明，这种自动定量分析方法对于研究细胞脂质代谢是有用的，并且有可能用于与动脉粥样硬化和脂质相关疾病相关的治疗剂的临床前高通量筛选。
