Bessel-Beam Hyperspectral CARS Microscopy with Sparse Sampling: Enabling High-Content High-Throughput Label-Free Quantitative Chemical Imaging
Bessel-Beam Hyperspectral CARS Microscopy with Sparse Sampling: Enabling High-Content High-Throughput Label-Free Quantitative Chemical Imaging
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DOI：
10.1021/acs.analchem.7b04039
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发表时间：
2018-03-20
影响因子：
7.4
通讯作者：
Borri, Paola
中科院分区：
文献类型：
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作者：
Masia, Francesco;Pope, Iestyn;Borri, Paola
基于显微镜的细胞系统的高含量和高通量分析在药物发现中起着核心作用。然而，对于对比度和特异性，大多数测定需要荧光读数，这总是伴随着真实生物条件改变的风险。在这项工作中，我们展示了一个无标记的成像平台，它结合了化学特定的高光谱相干反斯托克斯拉曼散射显微镜与稀疏采样和贝塞尔光束照明。这使我们能够高速筛选多孔板，同时保留高光谱成像的高含量化学分析。为了证明该方法的实用性，我们解决了药物筛选中的一个关键副作用，即药物诱导的肝组织内脂质储存。我们筛选了15种药物和中性脂质添加到人HepG2肝细胞中的组合，并开发了一种高含量的定量数据分析管道，该管道提取了脂质和蛋白质组分的光谱和空间分布。然后，我们使用它们的组合来训练支持向量机判别算法。在完全无标记的试验中实现了磷脂质病与脂肪变性方面的药物反应分类。
Microscopy-based high-content and high-throughput analysis of cellular systems plays a central role in drug discovery. However, for contrast and specificity, the majority of assays require a fluorescent readout which always comes with the risk of alteration of the true biological conditions. In this work, we demonstrate a label-free imaging platform which combines chemically specific hyperspectral coherent anti-Stokes Raman scattering microscopy with sparse sampling and Bessel beam illumination. This enabled us to screen multiwell plates at high speed, while retaining the high-content chemical analysis of hyperspectral imaging. To demonstrate the practical applicability of the method we addressed a critical side effect in drug screens, namely, drug-induced lipid storage within hepatic tissue. We screened 15 combinations of drugs and neutral lipids added to human HepG2 liver cells and developed a high-content quantitative data analysis pipeline which extracted the spectra and spatial distributions of lipid and protein components. We then used their combination to train a support vector machine discriminative algorithm. Classification of the drug responses in terms of phospholipidosis versus steatosis was achieved in a completely label-free assay.