The principles, strengths and limitations of several nonlinear optical (NLO) methods for characterizing biological systems are reviewed. NLO methods encompass a wide range of approaches that can be used for real-time, in-situ characterization of biological systems, typically in a label-free mode. Multiphoton excitation fluorescence (MPEF) is widely used for high-quality imaging based on electronic transitions, but lacks interface specificity. Second harmonic generation (SHG) is a parametric process that has all the virtues of the two-photon version of MPEF, yielding a signal at twice the frequency of the excitation light, which provides interface specificity. Both SHG and MPEF can provide images with high structural contrast, but they typically lack molecular or chemical specificity. Other NLO methods such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) can provide high-sensitivity imaging with chemical information since Raman active vibrations are probed. However, CARS and SRS lack interface and surface specificity. A NLO method that provides both interface/surface specificity as well as molecular specificity is vibrational sum frequency generation (SFG) spectroscopy. Vibration modes that are both Raman and IR active are probed in the SFG process, providing the molecular specificity. SFG, like SHG, is a parametric process, which provides the interface and surface specificity. SFG is typically done in the reflection mode from planar samples. This has yielded rich and detailed information about the molecular structure of biomaterial interfaces and biomolecules interacting with their surfaces. However, 2-D systems have limitations for understanding the interactions of biomolecules and interfaces in the 3-D biological environment. The recent advances made in instrumentation and analysis methods for sum frequency scattering (SFS) now present the opportunity for SFS to be used to directly study biological solutions. By detecting the scattering at angles away from the phase-matched direction even centrosymmetric structures that are isotropic (e.g., spherical nanoparticles functionalized with self-assembled monolayers or biomolecules) can be probed. Often a combination of multiple NLO methods or a combination of a NLO method with other spectroscopic methods is required to obtain a full understanding of the molecular structure and surface chemistry of biomaterials and the biomolecules that interact with them. Using the right combination methods provides a powerful approach for characterizing biological materials.

中文翻译：

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表征分子结构和表面化学的非线性光学方法。

综述了几种用于表征生物系统的非线性光学（NLO）方法的原理，优势和局限性。NLO方法涵盖了广泛的方法，这些方法通常可在无标签模式下用于生物系统的实时，原位表征。多光子激发荧光（MPEF）被广泛用于基于电子跃迁的高质量成像，但缺乏界面特异性。二次谐波生成（SHG）是一种参数化过程，具有MPEF双光子版本的所有优点，所产生的信号频率是激发光频率的两倍，从而提供了界面特异性。SHG和MPEF均可提供具有高结构对比度的图像，但它们通常缺乏分子或化学特异性。由于探测了拉曼主动振动，其他NLO方法（如相干反斯托克斯拉曼散射（CARS）和受激拉曼散射（SRS））可以提供具有化学信息的高灵敏度成像。但是，CARS和SRS缺乏界面和表面特异性。提供界面/表面特异性以及分子特异性的NLO方法是振动和频率生成（SFG）光谱。在SFG过程中探查同时具有拉曼和红外活性的振动模式，从而提供分子特异性。像SHG一样，SFG是一个参数化过程，它提供了界面和表面特异性。SFG通常是在平面样品的反射模式下完成的。这产生了有关生物材料界面和与其表面相互作用的生物分子的分子结构的丰富而详细的信息。但是，2-D系统在理解3-D生物环境中生物分子和界面之间的相互作用方面存在局限性。和频散射（SFS）的仪器和分析方法的最新进展为SFS直接用于研究生物溶液提供了机会。通过检测远离相位匹配方向的角度的散射，甚至可以探测到各向同性的中心对称结构（例如，用自组装单分子层或生物分子功能化的球形纳米粒子）。通常需要多种NLO方法的组合或NLO方法与其他光谱方法的组合，以全面了解生物材料及其相互作用的生物分子的分子结构和表面化学。使用正确的组合方法可为表征生物材料提供强大的方法。