Quantitative CARS Molecular Fingerprinting of Single Living Cells with the Use of the Maximum Entropy Method
Quantitative CARS Molecular Fingerprinting of Single Living Cells with the Use of the Maximum Entropy Method
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DOI：
10.1002/anie.201001560
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发表时间：
2010-01-01
影响因子：
16.6
通讯作者：
Hamaguchi, Hiro-o
中科院分区：
文献类型：
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作者：
Okuno, Masanari;Kano, Hideaki;Hamaguchi, Hiro-o
振动光谱通常被称为“分子指纹”。一旦获得了所谓的指纹区域（1800 cm ↑ [2]-800 cm ↑ [2]-1）的振动光谱，该区域显示出许多对分子结构高度敏感的骨架振动，我们就可以从任何种类的材料系统（从分子到活细胞）中获得详细的、否则无法获得的分子信息。由于生物系统是由分子组成的，振动光谱学在生命/生物科学中应该和在材料科学中一样有用。对于生物应用，拉曼光谱比红外光谱更适合，因为它不受水的严重影响，水在红外区具有非常强的吸收。在显微镜下的空间分辨实验中，拉曼光谱比红外光谱更有利。拉曼光谱可以通过使用共焦光学显微镜来实现亚微米的空间分辨率，但是红外光谱由于衍射极限而只能管理几微米的空间分辨率。因此，线性和非线性的拉曼光谱技术已广泛用于在显微镜下测量生物系统的振动光谱和图像。[1-6]其中，相干反斯托克斯拉曼散射（汽车）显微镜已经成为一种用于高速振动成像的聚焦技术。[1-3该技术采用一组两条窄带激光线（w1和w2）来激发孤立的强拉曼带（通常为CH伸缩带）以获得振动图像。然而，目前的汽车显微镜很难应用于指纹区域，其中许多拉曼谱带彼此非常接近。它是缺乏光谱覆盖率和分辨率，这是需要在拥挤的指纹region. In分离频带，我们扩展汽车显微镜汽车显微光谱的“光谱成像”，我们使用了一个简单的光谱分析，以提取定量的振动信息，从拥挤的振动光谱和映射出特定的分子部分内的细胞。我们使用多重汽车方法[10-13]（图1a）获得
Vibrational spectra are often called “molecular fingerprints”. Once vibrational spectra are obtained for the so-called fingerprint region (between 1800 cmÀ1 and 800 cmÀ1), which exhibits many skeletal vibrations that are highly sensitive to molecular structure, we can derive detailed and otherwise unobtainable molecular information from any kind of material systems ranging from a molecule to a living cell. Since biological systems are made up of molecules, vibrational spectroscopy should be useful in life/biological sciences as much as it is in material sciences. For biological applications, Raman spectroscopy is more suitable than infrared spectroscopy, because it is not seriously influenced by water, which has very strong absorption in the infrared region. Raman spectroscopy is more advantageous than infrared also in space-resolved experiments under a microscope. Raman spectroscopy can achieve sub-micrometer spatial resolution by using a confocal optical microscope but infrared can only manage spatial resolution of a few micrometers because of the diffraction limit. Thus, Raman spectroscopic techniques, both linear and non-linear, have been extensively used for measuring vibrational spectra and images of biological systems under a microscope.[1–6] Among these, coherent anti-Stokes Raman scattering (CARS) microscopy has been a technique of focus for high-speed vibrational imaging.[1–3, 7–9] This technique employs a set of two narrow-band laser lines (w1 and w2) for exciting an isolated and intense Raman band (generally the CH stretch band) to obtain a vibrational image. However, current CARS microscopy is difficult to apply to the fingerprint region, where many Raman bands are located very close to one another. It is lacking in spectral coverage and resolution, which are needed for separating bands in the congested fingerprint region.Herein, we extend CARS microscopy to CARS microspectroscopy by “spectral imaging”; we used a straightforward spectral analysis to extract quantitative vibrational information from congested vibrational spectra and map out specific molecular moieties within a cell. We used the multiplex CARS method [10–13](Figure1a) for obtaining