Fast vibrational imaging of single cells and tissues by stimulated Raman scattering microscopy.
Fast vibrational imaging of single cells and tissues by stimulated Raman scattering microscopy.
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DOI：
10.1021/ar400331q
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发表时间：
2014-08-19
影响因子：
18.3
通讯作者：
Cheng, Ji-Xin
中科院分区：
文献类型：
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作者：
Zhang, Delong;Wang, Ping;Slipchenko, Mikhail N.;Cheng, Ji-Xin
传统上，分子在试管中进行分析。以生物化学为例，我们关于细胞内容物的大部分知识来自使用免疫印迹和液相色谱-质谱等工具对固定细胞或组织匀浆进行的分析。这些工具可以指示分子的存在，但不能实时提供有关它们的位置或彼此相互作用的信息，限制了我们对所研究分子功能的理解。对于活细胞中标记分子的实时成像，荧光显微镜是首选工具。然而，荧光标记对于脂肪酸、氨基酸和胆固醇等小分子来说太大了。这些挑战凸显了开发允许原位或体内分子分析的化学成像平台的迫切需要。基于自发拉曼散射的振动光谱广泛用于细胞和组织中化学成分的无标记分析。然而，拉曼过程的效应较弱，限制了其在生命系统快速化学成像中的应用。相干拉曼散射显微镜具有高成像速度和 3D 空间分辨率，为生命系统中单细胞的实时振动成像提供了一种新方法。在大多数实验中，相干拉曼过程涉及两个激发场，分别表示为 ωp 处的泵浦和 ωs 处的斯托克斯。当泵浦场和斯托克斯场之间的跳动频率 (ωp – ωs) 与拉曼活性分子振动共振时，四个主要的相干拉曼散射过程同时发生，即 (ωp – ωs) + ωp 处的相干反斯托克斯拉曼散射 (CARS)、ωs – (ωp – ωs) 处的相干斯托克斯拉曼散射 (CSRS)、ωs 处的受激拉曼增益 (SRG) 和受激拉曼散射ωp 处的拉曼损耗 (SRL)。在 SRG 中，斯托克斯光束的强度有所增加，而在 SRL 中，泵浦光束的强度有所损失。 SRG和SRL都属于受激拉曼散射（SRS），其中泵浦场和斯托克斯场之间的能量差转移到分子以进行振动激发。 SRS 信号出现在与激发场相同的波长处，并且通常通过相敏检测方案来提取。由于拉曼跃迁而检测到的强度变化与 Im[χ(3)]IpIs 成正比，其中 χ(3) 代表三阶非线性磁化率，Ip 和 Is 代表泵浦场和斯托克斯场的强度。在本报告中，我们讨论了 SRS 显微镜技术开发和应用的最新进展。与 CARS 相比，SRS 对比度没有非共振背景。此外，SRS 强度与聚焦目标分子的密度成线性比例。对于单频成像，SRS 显微镜的速度比线扫描拉曼显微镜快约 1000 倍，比点扫描拉曼显微镜快 10 000 倍。需要强调的是，SRS 和自发拉曼散射是相辅相成的。自发拉曼光谱覆盖了分子振动的整个窗口，可以通过多变量分析提取微妙之处。 SRS 通过关注单个拉曼波段或目标分子的定义光谱窗口来提供速度优势。将单频 SRS 成像和自发拉曼光谱集成在一个平台上，可以对单个活细胞内的物体进行定量成分分析。
Traditionally, molecules are analyzed in a test tube. Taking biochemistry as an example, the majority of our knowledge about cellular content comes from analysis of fixed cells or tissue homogenates using tools such as immunoblotting and liquid chromatography–mass spectrometry. These tools can indicate the presence of molecules but do not provide information on their location or interaction with each other in real time, restricting our understanding of the functions of the molecule under study. For real-time imaging of labeled molecules in live cells, fluorescence microscopy is the tool of choice. Fluorescent labels, however, are too bulky for small molecules such as fatty acids, amino acids, and cholesterol. These challenges highlight a critical need for development of chemical imaging platforms that allow in situ or in vivo analysis of molecules. Vibrational spectroscopy based on spontaneous Raman scattering is widely used for label-free analysis of chemical content in cells and tissues. However, the Raman process is a weak effect, limiting its application for fast chemical imaging of a living system. With high imaging speed and 3D spatial resolution, coherent Raman scattering microscopy is enabling a new approach for real-time vibrational imaging of single cells in a living system. In most experiments, coherent Raman processes involve two excitation fields denoted as pump at ωp and Stokes at ωs. When the beating frequency between the pump and Stokes fields (ωp – ωs) is resonant with a Raman-active molecular vibration, four major coherent Raman scattering processes occur simultaneously, namely, coherent anti-Stokes Raman scattering (CARS) at (ωp – ωs) + ωp, coherent Stokes Raman scattering (CSRS) at ωs – (ωp – ωs), stimulated Raman gain (SRG) at ωs, and stimulated Raman loss (SRL) at ωp. In SRG, the Stokes beam experiences a gain in intensity, whereas in SRL, the pump beam experiences a loss. Both SRG and SRL belong to stimulated Raman scattering (SRS), in which the energy difference between the pump and Stokes fields is transferred to the molecule for vibrational excitation. The SRS signal appears at the same wavelengths as the excitation fields and is commonly extracted through a phase-sensitive detection scheme. The detected intensity change because of a Raman transition is proportional to Im[χ(3)]IpIs, where χ(3) represents the third-order nonlinear susceptibility, Ip and Is stand for the intensity of the pump and Stokes fields. In this Account, we discuss the most recent advances in the technical development and enabling applications of SRS microscopy. Compared to CARS, the SRS contrast is free of nonresonant background. Moreover, the SRS intensity is linearly proportional to the density of target molecules in focus. For single-frequency imaging, an SRS microscope offers a speed that is ∼1000 times faster than a line-scan Raman microscope and 10 000 times faster than a point-scan Raman microscope. It is important to emphasize that SRS and spontaneous Raman scattering are complementary to each other. Spontaneous Raman spectroscopy covers the entire window of molecular vibrations, which allows extraction of subtleties via multivariate analysis. SRS offers the speed advantage by focusing on either a single Raman band or a defined spectral window of target molecules. Integrating single-frequency SRS imaging and spontaneous Raman spectroscopy on a single platform allows quantitative compositional analysis of objects inside single live cells.
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影响因子：
2.5
作者：
Garbacik, Erik T.;Herek, Jennifer L.;Offerhaus, Herman L.
通讯作者：
Offerhaus, Herman L.
使用高光谱相干抗螺旋体拉曼散射显微镜的定量化学成像和无监督分析。
影响因子：
7.4
作者：
Masia, Francesco;Glen, Adam;Stephens, Phil;Borri, Paola;Langbein, Wolfgang
通讯作者：
Langbein, Wolfgang
影响因子：
3.3
作者：
Fu, Dan;Holtom, Gary;Freudiger, Christian;Zhang, Xu;Xie, Xiaoliang Sunney
通讯作者：
Xie, Xiaoliang Sunney
DOI：
10.1039/c3an02281a
发表时间：
2014-05-21
期刊：
The Analyst
影响因子：
--
作者：
Hu F;Wei L;Zheng C;Shen Y;Min W
通讯作者：
Min W
影响因子：
35
作者：
通讯作者：
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