Correction of optical distortions in dry depth profiling with confocal Raman microspectroscopy

We present a generalized approach to obtain improved Raman intensity profiles from in‐depth studies performed by confocal Raman microspectroscopy (CRM) with dry objectives. The approach is based on regularized deconvolution of the as‐measured confocal profile, through a kernel that simulates optical distortions due to diffraction, refraction and collection efficiency on the depth response. No specific shape or restrictions for the recovered profile are imposed. The strategy was tested by probing, under different instrumental conditions, a series of model planar interfaces, generated by the contact of polymeric films of well‐defined thickness with a substrate. Because of the aforementioned optical distortions, the as‐measured confocal response of the films appeared highly distorted and featureless. The signal computed after deconvolution recovers all the films features, matching very closely with the response expected. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of one‐dimensional spherically aberrated point spread function in depth profiling by confocal Raman microscopy

We present a simple experiment that allows the complete and direct characterization of the point spread function (PSF) in refraction‐aberrated depth profiling experiments with confocal Raman microscopy. We used a wedge‐shaped solid polymer film to induce refraction aberrations on the response of an infinitesimally thin Raman scatterer, represented by a polished silicon wafer. The system, with the film pasted on top of the Si wafer, was probed by a depth slicing technique under a dry‐optics configuration. Post‐acquisition processing of the Si and polymer intensity maps allowed the reconstruction of the axial PSF spatially resolved each 1 µm or less in the z‐axis and for virtually continuous values of focusing depth. In agreement with theory, we found that PSF broadens asymmetrically with focusing depth, with a marked shift in the focus point. From the shape of PSF, we obtained values of depth resolution within the film that confirm that axial discrimination is not drastically deteriorated, as suggested by previous works, and that confocal aperture effectively reduces the collection volume even under severe refraction interference. Copyright © 2013 John Wiley & Sons, Ltd.     

Laser-based volumetric colour-coded three-dimensional particle velocimetry

We present a method of three-dimensional particle velocimetry with a single digital colour camera using multiple colour illumination to encode image depth over a large volume. A copper vapour laser operating at 511 nm is used to pump an optical fibre producing a multiple-wavelength beam via multiple order stimulated Raman scattering. The beam is dispersed and formed into a stack of thin sheets to illuminate a volume of space. The spatial co-ordinates of particles imaged within the illuminated volume are obtained from their imaged x,y positions with depth discerned from particle hue (set by the wavelength of illumination). The method exhibits an RMS depth error of 3% in relation to the thickness of the illuminated region. This paper reports a proof-of-principle of three-dimensional particle imaging using a multi-wavelength laser source with a view to 3D-3C particle velocimetry.     

Confocal Raman microscopy: how to correct depth profiles considering diffraction and refraction effects

A new approach to obtain corrected depth profiles by confocal Raman microscopy, which considers diffraction and refraction effects is presented. The problem of diffraction effects encountered intrinsically in the confocal configuration can be described using a linear Fredholm integral equation of the first kind, which correlates apparent and true Raman intensities with the depth resolution curve of the instrument. Refractive index differences between air and the polymer sample, which cause further errors in the obtained depth profile due to strong aberration effects have been considered. This has been carried out using an empirical variation of the depth resolution function, which is able to simulate the broadening of the depth of focus with depth and also the discrepancy between nominal and measured depth scales. It is shown that considerable differences between apparent and corrected depth profiles exist at the surface and that these depend on the gradient of the profile and the depth resolution of the Raman microscope. Copyright © 2007 John Wiley & Sons, Ltd.     

Surface‐sensitive Raman microscopy with total internal reflection illumination

A Raman microscope using a total internal reflection (TIR) annular illumination geometry through a ZnSe solid immersion lens (SIL) is described. Spectra of a thin‐film sample of the transparent organic conductor poly(3,4‐ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) on a polyethylene terephthalate (PET) substrate are presented and compared with those from a conventional confocal Raman configuration. These spectra demonstrate a significant increase in surface selectivity upon the use of TIR illumination, as the decay length of the evanescent excitation field limits the depth of sample probed in this configuration. Spectral interference from the underlying PET substrate layer is thus greatly reduced. An increase in surface selectivity is also demonstrated for spectra acquired through the SIL with uniform illumination. Raman images of a micropatterned PEDOT:PSS film acquired with TIR illumination are also reported. Enhanced lateral resolution is realized in this configuration because of the immersion effect of the SIL, and the sampling depth is limited to 150 nm by the choice of illumination geometry. This results in analysis volumes on the order of tens of femtoliters, nearly two orders of magnitude smaller than typically achieved in conventional confocal Raman microscopes. This approach yields Raman spectra and images with surface selectivity significantly greater than can be achieved in confocal Raman, and provides a valuable tool for the microanalysis of thin surface films. Published in 2010 by John Wiley & Sons, Ltd.     

基于双边拟合的高稳定性共焦拉曼光谱定焦方法

共焦拉曼光谱技术可实现定量、无损、无需标记的样品微区“分子结构特征和物质组成信息”成像,被广泛应用于生物医学、物理化学以及材料科学等领域。由于共焦拉曼系统采用“点”激发和“点”探测的探测机制,且拉曼散射光谱信号微弱,导致成像所需时间可长达数小时甚至数十小时;测量过程中系统极易受环境变化、空气扰动等因素影响产生漂移,造成被测样品离焦,从而导致成像质量不稳定。针对现有共焦拉曼系统对样品定焦能力不足、样品易产生离焦误差、系统漂移大等问题,本文提出了一种基于双边拟合的高稳定性共焦拉曼光谱定焦方法。该方法首先对共焦拉曼光谱强度轴向响应曲线两侧对样品离焦敏感的数据区间分别进行线性拟合,得到两条拟合直线方程;然后,将所得的两条直线方程相减得到新的差分直线;最后,通过差分直线的过零点位置确定系统焦平面位置,实现了被测样品的高精度定焦,消除了离焦对系统测量结果的影响。以单晶硅表面同一位置,轴向扫描步距100 nm,进行60次重复定焦实验,实验获得的重复定焦极差为80.2 nm,说明系统具有良好的抗漂移能力。对周期5 μm的竖条栅格标准原子力台阶样品进行拉曼mapping成像测试,结果表明在长时间的成像过程中,和无定焦功能的图像相比,该方法获得的竖条栅格图像更清晰、边缘更锐利、信噪比较好。仿真分析和实验结果表明：提出的基于双边拟合共焦拉曼光谱探测方法可以提高系统的定焦准确度,抑制干扰因素导致的系统离焦对成像质量的影响,进而确保了系统探测的稳定性和成像分辨力,是一种自动定焦、抗漂移的拉曼光谱成像方法。     

一种快速测量共聚焦X射线分析装置探测微元尺寸方法

共聚焦X射线荧光技术是一种无损的三维光谱分析技术,在材料,生物,矿物样品分析,考古,证物溯源等领域具有广泛应用。共聚焦X射线荧光谱仪的核心部件为两个多毛细管X光透镜。一个为多毛细管X光会聚透镜（PFXRL）,其存在一后焦点,作用是把X光管所发出的发散X射线会聚成几十微米大小的高增益焦斑。另一透镜为多毛细管X光平行束透镜（PPXRL）,其存在一几十微米大小前焦点,置于X射线能量探测器前端,其作用是接收特定区域的X射线荧光信号。在共聚焦Ｘ射线荧光谱仪中,PFXRL的后焦点与PPXRL的前焦点重合,所形成的区域称作探测微元。只有置于探测微元区域的样品能够被谱仪检测到,使样品与探测微元相对移动,逐点扫描,便能够对样品进行三维无损的X射线分析。探测微元的尺寸决定共聚焦X射线荧光谱仪的空间分辨率,因此精确测量谱仪的探测微元的尺寸是非常重要的。如图1所示,谱仪探测微元可以近似为椭球体,其尺寸可以用水平方向分辨率X, Y,和深度分辨率Z表示。目前,常采用金属细丝或金属薄膜通过刀口扫描的方法测量谱仪探测微元尺寸。为了精确的从三个维度测量探测微元尺寸,金属细丝直径要小于探测微元尺寸。金属细丝和探测微元都是数十微米级别的尺寸大小,很难把金属靠近探测微元。为了得到探测微元在不同X射线能量下尺寸变化曲线,要采用多种金属细丝测量。采用单个金属细丝依次测量比较耗费时间。采用金属薄膜可以很方便地测量探测微元的深度分辨率Z,但是当测量水平分辨率X, Y时,难以准确测量。为了解决以上谱仪探测微元测量中存在的问题,本文提出采用多种金属丝平行粘贴在硬纸片上作为样品用于快速测量探测微元尺寸。附有金属细丝的硬纸片靠近谱仪探测微元,可以将探测微元置于硬纸片所在平面。由于硬纸片与金属细丝在同一水平面,在谱仪摄像头的协助下,可以把金属细丝迅速的靠近探测微元。靠近探测微元后,在全自动三维样品台的协助下,金属细丝沿两个方向对探测微元分别进行一次二维扫描。通过对二维扫描数据的处理便可以获得探测微元尺寸随入射X射线能量变化曲线。采用此方法对实验室所搭建的共聚焦X射线荧光谱仪的探测微元进行了测量。     

Specific effects and deconvolution in submicrometre EPMA: application to binary diffusion

Electron probe (x‐ray) microanalysis (EPMA) is nowadays a classical and well‐established method for qualitative and (semi)quantitative evaluation of the elemental composition of the (near) surface of a sample on the micrometre scale. This technique can be used to determine concentration profiles due to (inter)diffusion in materials at submicrometre resolution if physical and geometrical effects that occur during the measurement process are accounted for. Standard phenomena are usually corrected by commercial software for a homogeneous elemental composition in the analysed area. However, in the case of a diffusion process on a small scale, the composition is no longer homogeneous and the effect of the hemispherical volume of the x‐ray emission on the spatial resolution of the concentration profiles, and consequently on the diffusion coefficients, has to be considered. A radial x‐ray distribution associated with the classical depth distribution, ?(z), allows for the definition of a 2D x‐ray emission function for medium to heavy materials. This enables one to study the effect of some geometrical parameters on the measured concentration profile and to propose a method of reconstructing the real weight fraction profile from the measured profile of the x‐ray intensities by using regularized deconvolution algorithms. Copyright © 2003 John Wiley & Sons, Ltd.     

小尺寸样品CARS图像信号分布实验研究

通过设计实验与分析模型,研究相干反斯托克斯拉曼散射成像对于样品尺寸小于系统点扩展函数尺寸的情况,分析相干反斯托克斯拉曼散射图像周围的depth的形成原因。分析过程首次引入轴向传输动态位移光线（此光线由相干体积元内轴心光线抽象出）对球形或柱形样品直径小于系统点扩展函数横向与轴向尺寸进行建模解析,对于所建模型定量分析结果表明,对于样品尺寸小于系统点扩展函数尺寸样品折射率调制了有效作用深度。Gouy相移只是其表观现象,是一个伴随性特征,因为物理学中存在的另一个极端,当样品尺寸足够小且具有相当的折射率,Gouy相移的作用近似为0,此时,样品周围的depth主要与样品的折射率及系统相干层析体积元内有效作用长度有关,相干反斯托克斯拉曼散射参量信号,具有自身的波矢匹配条件,不仅有大小,有方向,而且还受到样品自身材料折射率的影响比较明显。也就是对于参量过程波矢匹配条件是因,Gouy相移是伴随性特征。自此,通过相干反斯托克斯拉曼散射间接说明所用的紧聚焦参量信号成像过程,正是因为波矢匹配条件的存在使二次信号继承了激发光的紧聚焦特性,而拥有了继承性的Gouy相移,结合实验结果采用物理学的极端假设表明,Gouy相移不是产生depth的主要原因,主要原因是样品及周围的环境的折射率与系统相干层析体积元内有效作用长度之间共同作用的结果。而实验结果与模型定量分析的结果相吻合,此实验帮我们找到了影响CARS图像样品周围depth的成因机制,对于其他小尺寸样品的参量成像结果的分析具有一定的借鉴意义。通过设计实验,结合定量模型分析,首次明确造成小尺寸样品CARS图像周围的depth的真正原因,此抽象模型的拓展性对纳观参量过程的分析具有得天独厚的优势。抽象出的主能量光线动态位移模型成功分析纳观成像结果表明,相干作用长度之内有效作用长度及其行进路径的过程论的主因分析方法是研究纳观作用机制的最佳方法。     

3D Raman mapping of uniaxially loaded 6H‐SiC crystals

Raman spectroscopy is used to investigate the three‐dimensional stress distribution in 6H‐silicon carbide (SiC) specimens subjected to stresses up to 3.7 GPa along the c‐axis. Specifically, the relative Raman shift of the longitudinal optic phonon of 6H‐SiC is used to evaluate the local stress across the bulk crystal. For this purpose, an anvil device with opposed 6H‐SiC and sapphire specimens was used. After subjecting the anvils to uniaxial load, several series of two‐dimensional Raman maps were registered at different depths in the 6H‐SiC anvil. The analysis of the Raman spectra reveals an exponential decay of the stress as a function of the depth. A novel phenomenological Grüneisen‐like model is introduced here to account for such observation. On the contrary, the in‐plane stress analysis shows a radial Gaussian‐like distribution regardless the depth, a distinct behavior that is attributed to the appearance of shear stress components. The suitability of both models and their applicability to other materials are discussed, along with some future directions. Copyright © 2013 John Wiley & Sons, Ltd.     

Optofluidic Raman sensor for simultaneous detection of the toxicity and quality of alcoholic beverages

We report a chemometric prediction of the toxicity and quality of liquor using an optofluidic sensor based upon Waveguide Confined Raman Spectroscopy (WCRS). The WCRS sensor was used to record the Raman spectra, each obtained from a 20 µl sample of a given alcoholic beverage with and acquisition time of 20 s. This was used to predict, simultaneously, both the methanol concentration (toxicity) and ethanol concentration (quality), with an accuracy of 0.1% and 0.7% by volume, respectively, using a Partial Least Squares‐based chemometric model. The model sensor is shown to be capable of identifying toxic liquors, based on the test performed on different types of liquor samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Lipid organization and stratum corneum thickness determined in vivo in human skin analyzing lipid–keratin peak (2820–3030 cm−1) using confocal Raman microscopy

While the intercellular lipid structure of the stratum corneum (SC) plays an important role in the skin barrier function, the depth‐dependent profile of the intercellular lipids contributes decisively to deepen the understanding of the skin barrier function, drug penetration, development of skin diseases and their medication. The depth‐dependent profile of the lipids' chemico‐physical properties, such as the solid–fluid phase transition and the order–disorder transition, can exclusively be measured in human skin in vivo by means of confocal Raman microscopy. In the present paper, the lipid–keratin peak (2820–3030 cm⁻¹) was investigated. The lipid‐related Raman peaks centered at 2850 cm⁻¹ and 2880 cm⁻¹ were deconvoluted using Gaussian functions and investigated for their depth‐dependent shape and positional changes. Different fitting procedures show that even an additional Gaussian function cannot be used to fully characterize the lipid's polymethylene chains around 2880 cm⁻¹, which justifies the introduction of the sharpness of the peak centered near 2880 cm⁻¹. The results show that the 2880 cm⁻¹ peak sharpness might be used for determining the SC thickness. The concentration of the lipids with long‐chain carbon backbone (free fatty acids and ceramides) semi‐quantitatively decreases from 10 µm to 20 µm (SC thickness is 19.8 µm). The maximum position and broadness of the Gaussian peak centered at 2850 cm⁻¹ show that near the surface and in the deeper layers of the SC, the state of the lipids is more fluid and disordered compared to the medium layers of the SC. Copyright © 2016 John Wiley & Sons, Ltd.     

Integration of correlative Raman microscopy in a dualbeam FIB SEM

We present an integrated confocal Raman microscope in a focused ion beam scanning electron microscope (FIB SEM). The integrated system enables correlative Raman and electron microscopic analysis combined with focused ion beam sample modification on the same sample location. This provides new opportunities, for example the combination of nanometer resolution with Raman advances the analysis of sub‐diffraction‐sized particles. Further direct Raman analysis of FIB engineered samples enables in situ investigation of sample changes. The Raman microscope is an add‐on module to the electron microscope. The optical objective is brought into the sample chamber, and the laser source, and spectrometer are placed in a module attached onto and outside the chamber. We demonstrate the integrated Raman FIB SEM function with several experiments. First, correlative Raman and electron microscopy is used for the investigation of (sub‐)micrometer‐sized crystals. Different crystals are identified with Raman, and in combination with SEM the spectral information is combined with structurally visible polymorphs and particle sizes. Analysis of sample changes made with the ion beam is performed on (1) structures milled in a silicon substrate and (2) after milling with the FIB on an organic polymer. Experiments demonstrate the new capabilities of an integrated correlative Raman–FIB–SEM. Copyright © 2016 John Wiley & Sons, Ltd.     

A large‐solid‐angle X‐ray Raman scattering spectrometer at ID20 of the European Synchrotron Radiation Facility

An end‐station for X‐ray Raman scattering spectroscopy at beamline ID20 of the European Synchrotron Radiation Facility is described. This end‐station is dedicated to the study of shallow core electronic excitations using non‐resonant inelastic X‐ray scattering. The spectrometer has 72 spherically bent analyzer crystals arranged in six modular groups of 12 analyzer crystals each for a combined maximum flexibility and large solid angle of detection. Each of the six analyzer modules houses one pixelated area detector allowing for X‐ray Raman scattering based imaging and efficient separation of the desired signal from the sample and spurious scattering from the often used complicated sample environments. This new end‐station provides an unprecedented instrument for X‐ray Raman scattering, which is a spectroscopic tool of great interest for the study of low‐energy X‐ray absorption spectra in materials under in situ conditions, such as in operando batteries and fuel cells, in situ catalytic reactions, and extreme pressure and temperature conditions.     

Raman of indigo on a silver surface. Raman and theoretical characterization of indigo deposited on silicon dioxide-coated and uncoated silver nanoparticles

Raman, surface-enhanced Raman scattering, and shell isolated nanoparticles-enhanced Raman scattering techniques were used to study the indigo–nanoparticle interaction nature. Silver nanoparticles were employed with and without a silicon dioxide spacer inert layer. The SERS spectral profile, obtained using silver nanoparticles, is different from the Raman one, which led to the proposition that the indigo–silver interaction is in the range of intermolecular interactions. SERS spectral reproducibility suggests identical organization and orientation of the analyte on the metal surface. The shell isolated nanoparticles enhanced Raman scattering spectrum of indigo, obtained by using silicon dioxide coated silver nanoparticles resulted similar to its Raman spectrum. This result indicates that the indigo structure is chemically unmodified by the silicon dioxide-coated silver surface. From the shell-isolated nanoparticles-enhanced Raman scattering experiments, the electromagnetic mechanism is proposed as the reason for the spectral enhancement. Theoretical calculations allow one to infer both the indigo–silver surface interaction nature and the orientation of indigo on the surface.     

微晶硅薄膜带隙态及微结构的研究

研究了氢化微晶硅薄膜费米能级以上的带隙态密度分布与薄膜微结构关系.采用拉曼谱和红外谱表征不同H稀释比制备的微晶硅薄膜的微结构.薄膜带隙态密度分布由调制光电流的相移分析技术测得.采用三相模型（非晶相、晶相和界面相）分析了薄膜带隙态密度与薄膜微结构的关系.结果表明,材料的带隙态密度随着界面相的增加而增加,当界面体积分数达到最大时,薄膜的带隙态密度也最大,即材料的带隙态密度与界面体积分数正相关. 关键词： 带隙态 界面相 微晶硅 调制光电流     

Highly ordered periodic array of metallic nanodots fabricated through template‐assisted nanopatterning and its use for surface‐enhanced Raman scattering spectroscopy of flexible crystalline‐silicon nanomembranes

We report on the fabrication of highly uniform periodic arrays of metallic nanodots formed on glass substrates through a facile anodic aluminum oxide template‐assisted process and their use in selective surface‐enhanced Raman scattering spectroscopy to probe the structural properties of flexible crystalline‐silicon nanomembranes deposited on polymer substrates. A 14‐fold enhancement in Raman scattering was observed from the thin flexible silicon membranes deposited on polyethylene terephthalate substrates, which were used to probe the internal strains in the silicon nanomembrane as a function of the bending radius. Copyright © 2011 John Wiley & Sons, Ltd.     

Topographically controlled growth of silver nanoparticle clusters

We present a straightforward method to form regular arrays of chemically synthesized Ag nanoparticle clusters, which are preferentially nucleated and developed inside pre‐patterned Si nanowells. The sizes and shapes of the clusters follow the dimensions of the lithographically defined Si nanowells, which here have a depth of 100 nm and form a square array with a pitch of 300 nm. Potential use of the array of Ag nanoparticle clusters as a surface‐enhanced Raman scattering (SERS) based sensing platform is demonstrated by the calculation of locally enhanced electromagnetic fields and by confocal microscopic measurements of increased Raman signals from organic molecules adsorbed on the Ag nanoparticle clusters. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Quantitative surface enhanced Raman scattering measurements at the early stage of active agent release processes

The surface enhanced Raman scattering spectroscopy (SERS) is introduced as a new method to probe the initial release of active agents from controlled delivery systems. As a model system, mitoxantrone‐loaded polypropylene specimens immersed in water have been utilized. Surface enhanced resonance Raman scattering (SERRS) measurements allowed the quantitative delineation of the initial drug release profile. SERRS was also compared in early stage release processes with UV–vis absorption often used in traditional quantitative analysis via HPLC, a common technique for controlled release evaluation. More and above the high selectivity of the Raman Effect, SERS has been proved as a highly sensitive method to quantitatively monitor the initial release of the medicine even at the very early stage of the delivery process; UV–vis absorbance was unable to respond accordingly. Copyright © 2012 John Wiley & Sons, Ltd.     

Confocal Raman microscopy for simultaneous monitoring of partitioning and disordering of tricyclic antidepressants in phospholipid vesicle membranes

Characterization of drug–membrane interactions is important in order to understand the mechanisms of action of drugs and to design more effective drugs and delivery vehicles. Raman spectra provide compositional and conformational information of drugs and lipid membranes, respectively, allowing membrane disordering effects and drug partitioning to be assessed. Traditional Raman spectroscopy and other widely used bioanalytical techniques such as differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) typically require high sample concentrations. Here, we describe how temperature‐controlled, optical‐trapping confocal Raman microscopy facilitates the analysis of drug–membrane interactions using micromolar concentrations of drug, while avoiding drug depletion from solution by working at even lower lipid concentrations. The potential for confocal Raman microscopy as an effective bioanalytical tool is illustrated using tricyclic antidepressants (TCAs), which are cationic amphiphilic molecules that bind to phospholipid membranes and influence lipid phase transitions. The interaction of these drugs with vesicle membranes of differing head‐group charge is investigated while varying the ring and side‐chain structure of the drug. Changes in membrane structure are observed in Raman bands that report intra‐ and intermolecular order versus temperature. The partitioning of drugs into the membrane can also be determined from the Raman scattering intensities. These results demonstrate the usefulness of confocal Raman microscopy for the analysis of drug–membrane systems at biologically relevant drug concentrations. Effective tools for monitoring drug–membrane interactions are crucial for rational design of new drugs. Copyright © 2009 John Wiley & Sons, Ltd.