拉曼光谱测量中的荧光抑制方法综述

拉曼光谱是物质的特征结构谱,但是在许多情况下,除了激发出拉曼散射光之外,还会激发出强度远大于拉曼散射的荧光,从而限制了拉曼光谱的应用。因此在拉曼检测中对荧光采取抑制措施是十分必要的。在过去的几十年里发展了多种荧光抑制方法,包括荧光淬灭剂法、光漂白法、红外/紫外激发法、偏振调制法、移频激发法、高频调制法、门控法、数值处理法、非线性效应法等。文中概括性地介绍了上述各种技术方法的原理,并扼要地分析比较了各自的性能特点。     

Multifunctional imaging of endogenous contrast by simultaneous nonlinear and optical coherence microscopy of thick tissues

A variety of high resolution optical microscopy techniques have been developed in recent years for basic and clinical studies of biological systems. We demonstrate a trimodal microscope combining optical coherence microscopy (OCM) with two forms of nonlinear microscopy, namely two-photon excited fluorescence (2PF) and second harmonic generation (SHG), for imaging turbid media. OCM combines the advantages of confocal detection and coherence gating for structural imaging in highly scattering tissues. Nonlinear microscopy enables the detection of biochemical species, such as elastin, NAD(P)H, and collagen. While 2PF arises from nonlinear excitation of fluorescent species, SHG is a form of nonlinear scattering observed in materials that lack a center of inversion symmetry, such as type I collagen. Characterization of the microscope showed nearly diffraction-limited spatial resolution in all modalities. Images were obtained in fish scales and excised human skin samples. The primary endogenous sources of contrast in the dermis were due to elastin autofluorescence and collagen SHG. Multimodal microscopy allows the simultaneous visualization of structural and functional information of biological systems.     

岛状纳米多孔金制备及其SERS特性研究

表面增强拉曼散射（surface-enhanced Raman scattering,SERS）技术将纳米结构材料和拉曼光谱相结合,解决了传统拉曼散射技术灵敏度低的问题,为痕量物质检测提供了新的技术手段。高SERS活性基底材料是将拉曼光谱应用到痕量物质检测中的关键。采用磁控溅射技术结合脱合金工艺在硅片上制备出岛状纳米多孔金SERS基底,该基底的孔隙尺寸和金韧带宽度的比值远小于传统纳米多孔金,有效地增强了金韧带之间的电磁耦合效应,表现出更强的局域电磁场。该基底的检测极限可达约10^?10 mol·L^?1,且SERS光谱相对强度与浓度呈现出较好的线性关系,动态响应范围可达3个数量级。同时该基底结构均匀、性能稳定,制备工艺具有良好的可重复性。     

Rapid detection of microorganisms with nanoparticles and electron microscopy

Rapid detection of microorganisms is highly desirable. A procedure has been developed based on interactions between gold nanoparticles and proteins of microorganisms (Escherichia coli, Rhodococcus rhodochrous, and Candida sp.) followed by scanning electron microscopy (SEM). The nanoparticle-cell interaction was confirmed by ultraviolet resonance Raman spectroscopy (UVRS) in the SEM focus. Cell suspensions in a buffer were interacted with gold nanoparticles (<10 nm in diameter) prepared from tetrachloroauric acid and sodium borohydride. Possible interference of elevated salt concentrations was eliminated by dialysis in deionized water. Small (10 microL) aliquots of cell-nanoparticle suspensions were dried on a silicon wafer and photographed under an SEM. Characteristic bacterial or yeast cell images in the micrographs indicated the actual presence of microorganisms in the suspension examined. This was further confirmed by UV resonance Raman spectroscopy.     

AFM characterization of biomolecules in physiological environment by an advanced nanofabricated probe

Many relevant questions in biology and medicine require both topography and chemical information with high spatial resolution. Several biological events that occur at the nanometer scale level need to be investigated in physiological conditions. In this regard Atomic Force Microscopy (AFM) is one of the most powerful tools for label‐free nanoscale characterization of biological samples in liquid environment. Recently, the coupling of Raman spectroscopy to scanning probe microscopies has opened new perspectives on this subject; however, the coupling of quality AFM spectroscopy with Raman spectroscopy in the same probe is not trivial. In this work we report about the AFM capabilities of an advanced high‐resolution probe that has been previously nanofabricated by our group for coupling with Raman spectroscopy applications. We investigate its use for liquid AFM measurements on biological model samples like lipid bilayers, amyloid fibrils, and titin proteins. We demonstrate topography resolution down to nanometer level, force measurement and stable imaging capability. We also discuss about its potential as nanoscale chemical probe in liquid phase. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

基于荧光显微拉曼光谱的细菌快速解耦检测方法

在细菌检测环境中往往存在多种活细菌、死菌以及非生物微颗粒,它们形态和大小相近且耦合寄居在一起,这使得普通的菌类检测方法在检测细菌的种类和含量时很难奏效。提出一种细菌快速解耦检测方法,该方法采用荧光微视技术与拉曼光谱分析技术相结合,利用荧光微视技术实现活菌与非生物微颗粒及死菌的解耦检测,并且实现活菌的定位;利用拉曼光谱技术对定位活菌进行数据采集并结合Ward聚类分析法构建各种活菌的解耦检测模型。实验证明所提出解耦方法的对活菌的解耦检测精度达到了94%以上,且检测时间仅为普通显微拉曼光谱法的1/9。适用于烧伤伤口细菌感染检测等要求检测精度高、速度快的场合。     

拉曼光谱技术在石油化工领域应用进展

拉曼散射是物质一种分子光谱,可以获取物质结构和官能团信息,已经用于物质的定性分析。本文就拉曼光谱技术在石油化工行业的应用进行简述。结合化学计量学,拉曼光谱技术已经用于石油产品组成分析、燃料（汽油、柴油、航煤和生物柴油）质量指标测定、输油管线油品监控以及化工产品和石油产品在线监控等领域。拉曼光谱技术具有结果准确、分析速度快、操作简单、不破坏样品、多参数、便携以及可现场分析等优点,将在石油化工领域得到进一步推广应用。     

Note: tip enhanced Raman spectroscopy with objective scanner on opaque samples

We report on 14 nm lateral resolution in tip-enhanced Raman spectroscopy mapping of carbon nanotubes with an experimental setup that has been designed for the analysis of opaque samples in confocal side-access through a novel piezo-driven objective scanner. The objective scanner allows for fast and stable laser-to-tip alignment and for the adjustment of the focus position with sub-wavelength precision to optimize the excitation of surface plasmons. It also offers the additional benefit of imaging the near-field generated Raman scattering at the gap between tip and sample as direct control of the tip enhancement.     

Optimizing use of the structural chemical analyser (variable pressure FESEM‐EDX raman spectroscopy) on micro‐size complex historical paintings characterization

The novel Structural Chemical Analyser (hyphenated Raman spectroscopy and scanning electron microscopy equipped with an X‐ray detector) is gaining popularity since it allows 3‐D morphological studies and elemental, molecular, structural and electronic analyses of a single complex micro‐sized sample without transfer between instruments. However, its full potential remains unexploited in painting heritage where simultaneous identification of inorganic and organic materials in paintings is critically yet unresolved. Despite benefits and drawbacks shown in literature, new challenges have to be faced analysing multifaceted paint specimens. SEM?Structural Chemical Analyser systems differ since they are fabricated ad hoc by request. As configuration influences the procedure to optimize analyses, likewise analytical protocols have to be designed ad hoc. This paper deals with the optimization of the analytical procedure of a Variable Pressure Field Emission scanning electron microscopy equipped with an X‐ray detector Raman spectroscopy system to analyse historical paint samples. We address essential parameters, technical challenges and limitations raised from analysing paint stratigraphies, archaeological samples and loose pigments. We show that accurate data interpretation requires comprehensive knowledge of factors affecting Raman spectra. We tackled: (i) the in‐FESEM?Raman spectroscopy analytical sequence, (ii) correlations between FESEM and Structural Chemical Analyser/laser analytical position, (iii) Raman signal intensity under different VP‐FESEM vacuum modes, (iv) carbon deposition on samples under FESEM low‐vacuum mode, (v) crystal nature and morphology, (vi) depth of focus and (vii) surface‐enhanced Raman scattering effect. We recommend careful planning of analysis strategies prior to research which, although time consuming, guarantees reliable results. The ultimate goal of this paper is to help to guide future users of a FESEM‐Structural Chemical Analyser system in order to increase applications.     

超燃发动机流场组分浓度的在线测量

为了正确评价超燃发动机试验状态,采用自发拉曼散射技术在线测量了超燃发动机流场的主要组分。基于发动机试验条件和发动机与光学诊断技术的接口,建立了用于发动机流场组分测量的自发拉曼散射实验系统;测量了多车次发动机试验过程中流场主要组分的拉曼光谱;最后,通过光谱计算获得了流场主要组分浓度信息并重点分析了来流氧气含量及其变化情况。实验显示:发动机试验中,部分车次试验补氧后的来流中氧气的最大含量达到了30%,最小含量为18%,说明发动机试验过程中,对补氧量的控制精确和稳定性还有待提高。结果表明:采用自发拉曼散射技术可以较好地完成来流主要组分浓度测量工作,测量结果可用于发动机试验数据的分析及来流补氧控制方式和控制精度的改进。     

Hyperspectral data influenced by sample matrix: the importance of building relevant reference spectral libraries to map materials of interest

Hyperspectral imaging (HSI) and mapping are increasingly used for visualization and identification of nanoparticles (NPs) in a variety of matrices, including aqueous suspensions and biological samples. Reference spectral libraries (RSLs) contain hyperspectral data collected from materials of known composition and are used to detect the known materials in experimental samples through a one‐to‐one pixel “mapping” process. In some HSI studies, RSLs created from raw NPs were used to map NPs in experimental samples in a different matrix; for example, RSLs created from NPs in suspension to map NPs in biological tissue. Others have utilized RSLs created from NPs in the same matrix. However, few studies have systematically compared hyperspectral data as a function of the matrix in which the NPs are found and its impact on mapping results. The objective of this study is to compare RSLs created from metal oxide NPs in aqueous suspensions to RSLs created from the same NPs in rat tissues following in vivo inhalation exposure, and to investigate the differences in mapping that result from the use of each RSL. Results demonstrate that the spectral profiles of these NPs are matrix dependent: RSLs created from NPs in positive control tissues mapped to experimental tissues more appropriately than RSLs created from NPs in suspension. Aqueous suspension RSLs mapped 0‐602 out of 500,424 pixels per tissue image while tissue RSLs mapped 689‐18,435 pixels for the same images. This study underscores the need for appropriate positive controls for the creation of RSLs for mapping NPs in experimental samples.     

Fundamental differences between micro- and nano-Raman spectroscopy

Electric field polarization orientations and gradients close to near-field scanning optical microscope (NSOM) probes render nano-Raman fundamentally different from micro-Raman spectroscopy. With x -polarized light incident through an NSOM aperture, transmitted light has x, y and z components allowing nano-Raman investigators to probe a variety of polarization configurations. In addition, the strong field gradients in the near-field of a NSOM probe lead to a breakdown of the assumption of micro-Raman spectroscopy that the field is constant over molecular dimensions. Thus, for nano-Raman spectroscopy with an NSOM, selection rules allow for the detection of active modes with intensity dependent on the field gradient. These modes can have similar activity as infra-red absorption modes. The mechanism can also explain the origin and intensity of some Raman modes observed in surface enhanced Raman spectroscopy.     

Near-field reflection backscattering apertureless optical microscopy: application to spectroscopy experiments on opaque samples, comparison between lock-in and digital photon counting detection techniques

An apertureless scanning near-field optical microscope (ASNOM) in reflection backscattering configuration is designed to conduct spectroscopic experiments on opaque samples constituted of latex beads. The ASNOM proposed takes advantage of the depth-discrimination properties of confocal microscopes to efficiently extract the near-field optical signal. Given their importance in a spectroscopic experiment, we systematically compare the lock-in and synchronous photon counting detection methods. Some results of Rayleigh's scattering in the near field of the test samples are used to illustrate the possibilities of this technique for reflection backscattering spectroscopy.     

基于柱矢量光束的纵向拉曼信号探测

提出了一种获得径向偏振光中纵向分量激发拉曼信号的方法,用于解决一般偏振拉曼信号无法检测分子取向垂直于样品的问题。首先使用聚焦后的角向偏振光和径向偏振光激发样品,然后用两种激发光产生的拉曼信号进行计算,得到由纯纵向光场激发的拉曼信号。使用Si(0 0 1)样品和Si(1 1 0)样品验证了该方法的可行性。这对于使用拉曼散射信号测量纵向取向的分子,以及纵向的分子振动模式具有一定意义。     

Invited review article: combining scanning probe microscopy with optical spectroscopy for applications in biology and materials science

This is a comprehensive review of the combination of scanning probe microscopy (SPM) with various optical spectroscopies, with a particular focus on Raman spectroscopy. Efforts to combine SPM with optical spectroscopy will be described, and the technical difficulties encountered will be examined. These efforts have so far focused mainly on the development of tip-enhanced Raman spectroscopy, a powerful technique to detect and image chemical signatures with single molecule sensitivity, which will be reviewed. Beyond tip-enhanced Raman spectroscopy and/or topography measurements, combinations of SPM with optical spectroscopy have a great potential in the characterization of structure and quantitative measurements of physical properties, such as mechanical, optical, or electrical properties, in delicate biological samples and nanomaterials. The different approaches to improve the spatial resolution, the chemical sensitivity, and the accuracy of physical properties measurements will be discussed. Applications of such combinations for the characterization of structure, defects, and physical properties in biology and materials science will be reviewed. Due to the versatility of SPM probes for the manipulation and characterization of small and/or delicate samples, this review will mainly focus on the apertureless techniques based on SPM probes.     

Combined in vivo multiphoton and CARS imaging of healthy and disease-affected human skin

We present combined epi-coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging with both chemical discrimination and subcellular resolution on human skin in vivo. The combination of both image modalities enables label-free imaging of the autofluorescence of endogenous fluorophores by two-photon excited fluorescence, as well as imaging of the distribution of intercellular lipids, topically applied substances and water by CARS. As an example for medical imaging, we investigated healthy and psoriasis-affected human skin with both image modalities in vivo and found indications for different lipid distributions on the cellular level.     

Polymers on graphite and gold: molecular images and substrate defects

Sub-monomolecular layers of a derivatized Phthalocyaninato-poly-siloxane (PCPS) and a Polydiacetylene (PDA) have been prepared on highly orientated pyrolytic graphite and gold by Langmuir-Blodgett and Langmuir-Schäfer techniques. Raman scattering and grazing-incidence-reflection infrared spectroscopy were used to characterize packing and molecular orientations within these films. It was found that PCPS forms similarly well ordered monolayers on both graphite and gold while PDA does not. Scanning tunnelling microscopy (STM) was performed in air using both the constant height and constant current mode. On highly orientated pyrolytic graphite substrates various defect structures based on a √3X√3 R 30° superstructure were found near localized defects and small graphite steps. They were carefully distinguished from molecular images of densely packed hydrocarbon chains. Defect rich graphite was characterized with both STM and Raman spectroscopy. Similarly, disordered and graphite-like regions were found on carbon fibres. The sensitivity of STM to surface defects in graphitic material turned out to be large compared to that of the Raman spectroscopic method. STM images of PCPS monolayers on graphite and gold exhibited parallel polymer rods, 2 nm apart from each other.     

A low-wavenumber-extended confocal Raman microscope with very high laser excitation line discrimination

We describe the simple modification of a confocal Raman imaging microscope to incorporate two ultra-narrow holographic notch filters. The modified microscope rejects the laser excitation line (Rayleigh peak) by a discrimination factor of ～10(11) and allows simultaneous measurements of Stokes/anti-Stokes Raman shifts as close as ～10/20 cm(-1) to the Rayleigh line. The extremely high rejection ratio of the Rayleigh peak results in its intensity becoming comparable to typical Raman scattering signals. This is essential for micro-Raman spectroscopy and imaging in the low-wavenumber region. We illustrate the resulting performance with measurements on silicon/silica, sapphire, sulfur, L-cystine, as well as on single-walled carbon nanotubes (SWNTs). We find that both aggregated (bulk) and individual (deposited on substrate) SWNTs demonstrate strong and broad characteristic Raman features below ～100 cm(-1)-in a region which has remained essentially unexplored in measurements of bulk SWNT samples and which has so far been inaccessible for Raman spectroscopy of individual SWNTs.     

Probing nanoscale surface enhanced Raman-scattering fluctuation dynamics using correlated AFM and confocal ultramicroscopy

We have studied the laser-excitation-intensity-dependent and Ag-nanocluster interstitial-site-dependent SERS intensity fluctuations under low molecule surface coverage of rhodamine 6G and cytochrome c. A new two-channel photon time-stamping system coupled with atomic force microscopic (AFM), Raman spectroscopic, and imaging microscopy was developed and applied to record Raman intensity fluctuation trajectories at sub-microsecond resolution correlated with in situ characterization of the nanoparticle clusters. Our experimental results suggest that the nanoconfinement of the local electromagnetic-field enhancement and the interaction of the local field with the molecules, presumably under rotational motions, result in nano-Raman fluctuations. The SERS spectral fluctuation was pertinent to the nanoscale local enhancement and local interaction of the molecules with the surface when the surface coverage of the nanoparticles was less than a monolayer, and the nanoscale interstitial space controlled the finite number of molecules to contribute the microscopic Raman signal collected from a diffraction-limited focus spot. The fluctuation amplitude significantly decreased with the number of molecules confined at the nanolocal field. The nano-SERS fluctuation dynamics were both photo-induced and spontaneous for rhodamine 6G, but only the photo-induced component was observable for cytochrome c. The fluctuation dynamics were also found to be highly inhomogeneous at interstitial sites with heterogeneous geometries. To interpret the observed nano-SERS fluctuation dynamics, we used computer simulation of optical multiple scattering, based on multi-sphere scattering Mie theory, and rotational diffusion of molecules at an interstitial site, based on a random walk in orientation space.     

基于远程拉曼光谱的物质检测研究

设计和建立轻型远程拉曼光谱探测系统,通过时域脉冲的发射源和数字延时电路实现拉曼信号同步采集,同时采用入瞳口径为50mm的伽利略望远光路增强拉曼信号的收集效率,并对易爆物质进行3m距离检测为例进行研究。其结果显示,系统具有非常高的信噪比、高分辨锐利的特征峰和快速检测能力,能应用于危险环境的识别和预警等方面。