Infrared, Raman and Visible Reflectance Spectroscopic Imaging

得到的结果说明了尖端增强拉曼光谱(TERS)和成像在一种新的顶部照明几何结构中的应用。径向偏振光束被用来产生沿光束传播方向、垂直于表面的电场分量，与线偏振光束相比，获得了五倍的增强。这种倍增增强有助于区分近场信号和远场拉曼背景。顶部照明配置允许应用TERS来研究各种表面的分子，例如金、玻璃和硅。研究了包括单壁碳纳米管在内的多种材料的近场拉曼光谱。获得了足够的增强，以允许获得不同直径的大束碳纳米管的表面分布的亚衍射分辨率的高光谱拉曼成像立方体。为了进一步促进复杂生物样品的纳米粒子增强拉曼成像，利用背散射显微镜的配置，探索了使用径向和方位偏振聚焦在金纳米粒子AFM尖端的更高阶模的使用。特别是，径向偏振配置提供了增强的空间分辨率。这是金属纳米粒子中感应电子振荡方向的结果，该方向由激光焦点处的电磁场定向。具体地说，电磁增强的方向性是至关重要的，这样被观察的分子在纳米结构尖端的顶端经历了最大的增强场。这些结果表明，TERS有可能在纳米尺度上提供分子分布的洞察力，例如，那些代表疾病状态的系统。 在优化与TERS相关的实验参数方面已经花费了大量的努力。虽然用647 nm激光激发的镀金AFM玻璃尖端在碳纳米管上观察到了明显的尖端增强，但为了对生物样品进行拉曼光谱解析，必须有更强的增强因子。例如，研究发现，与镀金的AFM玻璃尖端相比，附金颗粒球的玻璃微吸管提供了更大的增强因子。此外，为了观察和验证TERS效应，必须正确区分近场和主要的远场拉曼散射。在模型类脂膜的情况下，通过使用Langmuir-Blogett(LB)膜沉积技术在受控的层数中制备类脂膜来最小化远场拉曼散射。此外，LB膜沉积提供了一种保持针尖-衬底间隙的方法，这是针尖增强所需的。同样，利用囊泡融合的方法也可以制备薄层沉积的脂膜。囊泡融合沉积特别适合于将相对不溶的蛋白质结合到脂膜中。 而石英和云母衬底为脂肪沉积和形貌可视化提供了平坦的表面。通过选择有利于表面等离子激元在尖端激光激发时局域化的衬底，可以获得更大的尖端增强效应。利用金颗粒球微吸管，在金衬底上沉积的单一脂单分子膜上，在1秒的时间内观察到1800-200 cm-1区域的尖端增强拉曼散射。为了获得合适的样品原子力显微镜(AFM)形貌，必须在样品沉积之前对金衬底进行退火热处理，以将表面粗糙度从&gt；10A0降低到3-4A0。或者，从超平的表面剥离出来的金矿，如硅或云母，也提供了一致的地形。我们注意到黄金底物对脂膜形成的影响，因为脂类在镀金底物上形成的双层似乎不同于云母底物。 虽然红外光谱已被广泛用于化学表征，但红外光谱在研究样品表面不同距离的化学变化方面的真正共聚焦应用还没有得到证实。(尽管共聚焦表征在生物医学研究中是必不可少的，但主要局限于使用荧光和拉曼光谱技术进行结构分析。)因此，我们最近概述了一种新的红外光谱测量设计，尽管考虑了衍射限制，但z方向的理论空间分辨率为1 nm。我们的方法包括对时间扫描红外显微镜进行改造，增加一个高速、高精度的纳米定位器。为了调制光谱信号，纳米定位器以固定的周期调制物镜和样品之间的距离。这代表了傅里叶变换红外差示光谱技术的新应用。实验是从交替的HfO2和Al_2O_3原子层样品开始的，这些样品很有用，因为它们的化学成分很简单。蛋白质膜将被逐层组装，以研究单个双层内的光谱变化。

Results were obtained which illustrated the use of tip-enhance Raman spectroscopy (TERS) and imaging in a novel top illumination geometry. A radially polarized beam was used to generate an electric field component in the direction of beam propagation, normal to the surface, resulting in a five times enhancement compared to a linearly polarized beam. This multiplicative enhancement facilitates a discrimination of the near-field signal from the far-field Raman background. The top illumination configuration allows the application of TERS for investigating molecules on a variety of surfaces, such as gold, glass and silicon. The near field Raman spectra of a variety of materials, including single wall carbon nanotubes, were examined. Sufficient enhancement was obtained to permit the obtaining of a sub-diffraction resolution hyperspectral Raman imaging cube of the surface distribution of large bundles of carbon nanotubes of varying diameter. To further facilitate nanoparticle enhanced Raman imaging of complex biological specimens, the use of higher laser order modes with radial and azimuthal polarizations focused onto a gold nanoparticle AFM tip was explored using a back-scattering microscope configuration. In particular, the radial polarized configuration provided enhanced spatial resolution. This is a consequence of the direction of the induced electron oscillation in the metal nanoparticle, which is oriented by the electromagnetic field at the laser focus. Specifically, the directionality of the electromagnetic enhancement is critical such that the molecule under observation experiences the greatest enhanced field at the apex of the nanostructure tip. These results indicate the potential for TERS to provide insights in molecular distributions on the nanoscale, as, for example, those systems representative of diseased states. Significant effort has been expended in optimizing experimental parameters associated with TERS. Although tip-enhancement was clearly observed on carbon nanotubes using gold-coated AFM glass tips with 647nm laser excitation, a stronger enhancement factor is necessarily desired for the Raman spectroscopic elucidation of biological samples. For example, glass micropipettes with gold particle balls attached were found to provide greater enhancement factors than gold-coated AFM glass tips. Additionally, proper discrimination of the near-field Raman scattering from the predominant far-field Raman scattering is necessary for the observation and validation of TERS effect. In the case of model lipid membranes, far-field Raman scattering is minimized by preparing lipid membranes in controlled numbers of layers using the Langmuir-Blogett (LB) film deposition technique. Further, LB film deposition provides a mean to maintain the tip-substrate gap required for tip enhancement. Similarly, thin layer deposition of lipid membranes can also be prepared using vesicle fusion method. Vesicle fusion deposition is particularly suitable for incorporating proteins that are relatively insoluble into lipid membranes. While quartz and mica substrates provide flat surfaces for lipid deposition and for topography visualization. Greater tip-enhancement effect is achieved by choosing substrates that favor the localization of surface plasmons upon laser excitation at the tip apex. With a gold particle ball micropipette, tip-enhanced Raman scattering in the 1800-200 cm-1 region is observed in as little as 1 second acquisition time on a single lipid monolayer deposited on a gold substrate. To obtain proper atomic force microscopy (AFM) topography of the sample, the gold substrate must be annealed prior to sample deposition to reduce the surface roughness from >10A0 to 3-4A0. Alternatively, gold deposits stripped from an ultraflat surface, such as silicon or mica, also provides consistent topography. We note the effect of a gold substrate on lipid membrane formation as lipids appear to form bilayers differently on gold coated substrates than mica substrates. Although infrared (IR) spectroscopy has been widely used for chemical characterization, a true confocal application of IR spectroscopy for studying the variation in chemistry at different distances from the sample surface has not been demonstrated. (Confocal characterization, even though it is essential in biomedical studies, has been mostly limited to structural analyses using fluorescence and Raman spectroscopic techniques.) Thus, we have recently outlined a new design for IR spectroscopic measurements with a theoretical spatial resolution of 1nm in the z-direction despite diffraction limit considerations. Our approach involves the modification of a time-scanned IR-microscope with the addition of a high-speed, high-precision nano-positioner.. The distance between the objective and the sample is modulated by the nano-positioner at a fixed periodicity in order to modulate the spectral signal. This represents a new application of a Fourier-transform IR difference spectroscopic technique. Experiments are initiated with samples of alternating atomic layers of HfO2 and Al2O3, useful samples for their simple chemical composition. Protein membranes will be assembled, layer-by-layer, to study the spectroscopic variations within individual bilayers.