Optimisation of multimodal coherent anti-Stokes Raman scattering microscopy for the detection of isotope-labelled molecules
Optimisation of multimodal coherent anti-Stokes Raman scattering microscopy for the detection of isotope-labelled molecules
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DOI：
10.1117/12.2509280
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发表时间：
2019-03
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通讯作者：
Dale Boorman;I. Pope;W. Langbein;S. Hood;P. Borri;P. Watson
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作者：
Dale Boorman;I. Pope;W. Langbein;S. Hood;P. Borri;P. Watson
相干反斯托克斯拉曼散射（CARS）显微镜利用分子的固有振动共振来驱动光的非弹性散射，从而消除了外源荧光标记的需要，同时提供具有化学特异性的高分辨率三维图像。用氘取代氢原子是一种标记策略，它对化合物结构的改变最小，但由于CH2峰被诱导降移到拉曼光谱的一个区域，而该区域不包含其他化学物质的贡献，因此与其他细胞成分形成对比，因此与CARS兼容。我们介绍了我们的工作，使用氘化油酸来优化内部开发的多模态，多光子，激光扫描显微镜的设置，以精确识别拉曼光谱沉默区域内的碳-氘相关峰。应用数据分析程序，分解成化学成分的敏感性和浓度（FSC3），可以在高光谱CARS图像中识别和定量确定特定氘化化学成分的空间分辨率。从与氘化或非氘化油酸孵养的HeLa细胞中获得了完整的高光谱CARS数据集，随后的FSC3分析能够根据细胞的化学背景识别外源性氘化脂质在细胞内的位置。通过FSC3分析的应用，氘标记可以为小分子成像提供一种强大的技术，这种技术不适合传统的荧光技术。
Coherent anti-Stokes Raman scattering (CARS) microscopy utilises intrinsic vibrational resonances of molecules to drive inelastic scattering of light, and thus eradicates the need for exogenous fluorescent labelling, whilst providing high-resolution three-dimensional images with chemical specificity. Replacement of hydrogen atoms with deuterium presents a labelling strategy that introduces minimal change to compound structure yet is compatible with CARS due to an induced down-shift of the CH2 peak into a region of the Raman spectrum which does not contain contributions from other chemical species, thus giving contrast against other cellular components. We present our work using deuterated oleic acid to optimise setup of an in-house-developed multimodal, multiphoton, laser-scanning microscope for precise identification of carbon-deuterium-associated peaks within the silent region of the Raman spectrum. Application of the data analysis procedure, factorisation into susceptibilities and concentrations of chemical components (FSC3), enables the identification and quantitative spatial resolution of specific deuterated chemical components within a hyperspectral CARS image. Full hyperspectral CARS datasets were acquired from HeLa cells incubated with either deuterated or non-deuterated oleic acid, and subsequent FSC3 analysis enabled identification of the intracellular location of the exogenously applied deuterated lipid against the chemical background of the cell. Through application of FSC3 analysis, deuterium-labelling may provide a powerful technique for imaging small molecules which are poorly suited to conventional fluorescence techniques.