Nanoscale Chemical Imaging Using Tip‐Enhanced Raman Spectroscopy: A Critical Review

Methods for chemical analysis at the nanometer scale are crucial for understanding and characterizing nanostructures of modern materials and biological systems. Tip‐enhanced Raman spectroscopy (TERS) combines the chemical information provided by Raman spectroscopy with the signal enhancement known from surface‐enhanced Raman scattering (SERS) and the high spatial resolution of atomic force microscopy (AFM) or scanning tunneling microscopy (STM). A metallic or metallized tip is illuminated by a focused laser beam and the resulting strongly enhanced electromagnetic field at the tip apex acts as a highly confined light source for Raman spectroscopic measurements. This Review focuses on the prerequisites for the efficient coupling of light to the tip as well as the shortcomings and pitfalls that have to be considered for TERS imaging, a fascinating but still challenging way to look at the nanoworld. Finally, examples from recent publications have been selected to demonstrate the potential of this technique for chemical imaging with a spatial resolution of approximately 10 nm and sensitivity down to the single‐molecule level for applications ranging from materials sciences to life sciences.     

Imaging Nanometre‐Sized Hot Spots on Smooth Au Films with High‐Resolution Tip‐Enhanced Luminescence and Raman Near‐Field Optical Microscopy

A novel near‐field optical microscope based on a parabolic mirror is used for recording high‐resolution tip‐enhanced photoluminescence (PL) and Raman images with unprecedented sensitivity and contrast. The measurements reveal small islands on the Au surface with dimensions of only a few nanometres with locally enhanced Au PL. These islands appear as nanometre‐sized hot spots in tip‐enhanced Raman microscopy when benzotriazole molecules adsorbed on the Au surface serve as local sensors for the optical field. The spectra show that localized plasmons are the cause of both the locally enhanced Au PL and enhanced Raman scattering. This finding suggests that the dispersive background in the surface‐enhanced Raman spectra can be explained simply by the enhanced Au PL in the gap. Furthermore, our results show that the surface flatness must be better than 1 nm, to provide an optically homogeneous substrate for near‐field enhanced PL and Raman spectroscopy.     

Surface Enhanced Raman Imaging of Adsorbate-Covered SERS Active Gold Nano-Particles

Surface enhanced Raman imaging (SERI), the combination of surface enhanced Raman scattering (SERS) and micro-Raman imaging, has recently been developed as a surface imaging technique. It offers the high sensitivity together with chemical information at high two-dimensional spatial resolution. For example, Yang et al. have reported the study of SERI distribution on a roughened silver electrode in two-dimension. Recently a particular interesting application of this technique in our lab was to image the gold nano-particles, which were deposited on glassy carbon (GC) surfaces.     

The many facets of Raman spectroscopy for biomedical analysis

A critical review is presented on the use of linear and nonlinear Raman microspectroscopy in biomedical diagnostics of bacteria, cells, and tissues. This contribution is combined with an overview of the achievements of our research group. Linear Raman spectroscopy offers a wealth of chemical and molecular information. Its routine clinical application poses a challenge due to relatively weak signal intensities and confounding overlapping effects. Nonlinear variants of Raman spectroscopy such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) have been recognized as tools for rapid image acquisition. Imaging applications benefit from the fact that contrast is based on the chemical composition and molecular structures in a label-free and nondestructive manner. Although not label-free, surface enhanced Raman scattering (SERS) has also been recognized as a complementary biomedical tool to increase sensitivity. The current state of the art is evaluated, illustrative examples are given, future developments are pointed out, and important reviews and references from the current literature are selected. The topics are identification of bacteria and single cells, imaging of single cells, Raman activated cell sorting, diagnosis of tissue sections, fiber optic Raman spectroscopy, and progress in coherent Raman scattering in tissue diagnosis. The roles of networks—such as Raman4clinics and CLIRSPEC on a European level—and early adopters in the translation, dissemination, and validation of new methods are discussed.     

Label‐Free Molecular Imaging of Biological Cells and Tissues by Linear and Nonlinear Raman Spectroscopic Approaches

Raman spectroscopy is an emerging technique in bioanalysis and imaging of biomaterials owing to its unique capability of generating spectroscopic fingerprints. Imaging cells and tissues by Raman microspectroscopy represents a nondestructive and label‐free approach. All components of cells or tissues contribute to the Raman signals, giving rise to complex spectral signatures. Resonance Raman scattering and surface‐enhanced Raman scattering can be used to enhance the signals and reduce the spectral complexity. Raman‐active labels can be introduced to increase specificity and multimodality. In addition, nonlinear coherent Raman scattering methods offer higher sensitivities, which enable the rapid imaging of larger sampling areas. Finally, fiber‐based imaging techniques pave the way towards in vivo applications of Raman spectroscopy. This Review summarizes the basic principles behind medical Raman imaging and its progress since 2012.     

Raman Imaging: An Impending Approach Towards Cancer Diagnosis

In accordance with the recent studies, Raman spectroscopy is well experimented as a highly sensitive analytical and imaging technique in biomedical research, mainly for various disease diagnosis including cancer. In comparison with other imaging modalities, Raman spectroscopy facilitate numerous assistances owing to its low background signal, immense spatial resolution, high chemical specificity, multiplexing capability, excellent photo stability and non-invasive detection capability. In cancer diagnosis Raman imaging intervened as a promising investigative tool to provide molecular level information to differentiate the cancerous vs non-cancerous cells, tissues and even in body fluids. Anciently, spontaneous Raman scattering is very feeble due to its low signal intensity and long acquisition time but new advanced techniques like coherent Raman scattering (CRS) and surface enhanced Raman scattering (SERS) gradually superseded these issues. So, the present review focuses on the recent developments and applications of Raman spectroscopy-based imaging techniques for cancer diagnosis.     

亮氨酸与异亮氨酸的表面增强拉曼光谱

报道了在蛋白质氨基酸中唯一一对异构体氨基酸——亮氨酸和异亮氨酸的FT-拉曼光谱和在银胶基底上的表面增强拉曼光谱(SERS). 归属了各振动、增强峰位并分析了异构体氨基酸分子内不同振动模式引起的拉曼位移及其在不同pH值下SERS的变化. 分子内不同的振动模式主要源于异构体氨基酸中一个甲基和主链的不同连接次序, 表现在拉曼光谱; 亮氨酸的甲基摇摆ρ(CH₃)和非对称变形δ_as(CH₃)在962, 945, 924和1454, 1408 cm^－1; 异亮氨酸的ρ(CH₃), δ_as(CH₃)在922和1448, 1420, 1394 cm^－1. C—CO, C—C, H—O…H及骨架晶格振动峰位基本对应. 饱和液态的拉曼光谱和SERS中, 各基团振动峰位的差异表现得更为明显. 初步推测了这对氨基酸异构体在银表面吸附状态的模型.     

Bioorthogonal SERS Nanoprobes for Mulitplex Spectroscopic Detection,Tumor Cell Targeting,and Tissue Imaging

A surface‐enhanced Raman scattering (SERS) technique shows extraordinary features for a range of biological and biomedical applications. Herein, a series of novel bioorthogonal SERS nanoprobes were constructed with Gold nanoflower (AuNF) and Raman reporters, the signals of which were located in a Raman‐silent region of biological samples. AS1411 aptamer was also co‐conjugated with AuNF through a self‐assembled monolayer coverage strategy. Multiplex SERS imaging using these nanoprobes with three different bioorthogonal small‐molecule Raman reporters is successfully achieved with high multiplexing capacity in a biologically Raman‐silent region. These Raman nanoprobes co‐conjugated with AS1411 showed high affinity for tumor cells with overexpressed nucleolin and can be used for selective tumor cell screening and tissue imaging.     

Characterization of genuine and fake artesunate anti-malarial tablets using Fourier transform infrared imaging and spatially offset Raman spectroscopy through blister packs

In support of the efforts to combat the illegal sale and distribution of counterfeit anti-malarial drugs, we evaluated a new analytical approach for the characterization and fast screening of fake and genuine artesunate tablets using a combination of Raman spectroscopy, Spatially Offset Raman Spectroscopy (SORS) and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) imaging. Vibrational spectroscopy provided chemically specific information on the composition of the tablets; the complementary nature of Raman scattering and FTIR imaging allowed the characterization of both the overall and surface composition of the tablets. The depth-resolving power of the SORS approach provided chemically specific information on the overall composition of the tablets, non-invasively, through a variety of packaging types. Spatial imaging of the tablet surface (using ATR-FTIR) identified the location of domains of excipients and active ingredients with high sensitivity and enhanced spatial resolution. The advantages provided by a combination of SORS and ATR-FTIR imaging in this context confirm its potential for inclusion in the analytical protocol for forensic investigation of counterfeit medicines.     

Conjugated Polymer with Intrinsic Alkyne Units for Synergistically Enhanced Raman Imaging in Living Cells

Development of Raman‐active materials with enhanced and distinctive Raman vibrations in the Raman‐silent region (1800–2800 cm⁻¹) is highly required for specific molecular imaging of living cells with high spatial resolution. Herein, water‐soluble cationic conjugated polymers (CCPs), poly(phenylene ethynylene) (PPE) derivatives, are explored for use as alkyne‐state‐dependent Raman probes for living cell imaging due to synergetic enhancement effect of alkyne vibrations in Raman‐silent region compared to alkyne‐containing small molecules. The enhanced alkyne signals result from the integration of alkyne groups into the rigid backbone and the delocalized π‐conjugated structure. PPE‐based conjugated polymer nanoparticles (CPNs) were also prepared as Raman‐responsive nanomaterials for distinct imaging application. This work opens a new way into the development of conjugated polymer materials for enhanced Raman imaging.     

基于聚苯乙烯微球的拉曼增强效应及其应用于金单晶表面单层分子的检测

利用在样品表面上组装聚苯乙烯微球, 可以使得表面拉曼信号得到增强. 系统考察了增强效应与微球粒子尺寸的依赖关系, 发现当微球直径为3.00 μm 时, 拉曼信号的增强效应最强, 可以达到约5倍的增强. 进一步利用聚苯乙烯微球的增强效应, 获得了单层吸附在Au(111)表面上具有共振增强效应的异氰基孔雀石绿分子的拉曼信号, 得到约20倍的信号净增强, 相当于约3个数量级的拉曼增强效应, 表明利用这种方法可以显著提高单晶表面吸附分子的检测灵敏度. 这种增强效应主要是由于激光在透明微球的作用下, 在微球底部产生纳米光束流, 从而形成高度局域化的电磁场, 使拉曼散射过程得到极大的增强. 初步探讨了两种类型样品表面获得不同的增强效应的原因.     

Enhanced Raman Scattering by Polystyrene Microspheres and Application for Detecting Molecules Adsorbed on Au Single Crystal Surface

By assembling polystyrene microspheres on a sample surface, the surface Raman signal could be enhanced. The dependence of the enhancement effect on the size of microspheres was systematically investigated and it was found that microspheres with a diameter of 3.00 μm showed the highest enhancement of ca 5 folds. By utilizing the enhancement effect of the microspheres, the surface Raman intensity of malachite green isothiocyanate (MGITC) adsorbed on Au(111) surface could be enhanced by 20 folds, indicating that this method could effectively improve the detection sensitivity of surface Raman spectroscopy for the adsorbed species on single crystal surface. The later signal increment corresponds to the Raman enhancement effect of nearly 3 orders of magnitude. The enhancement effect is mainly owing to the formation of nanojets when a laser is focused on the microspheres of appropriate diameter. The formation of nanojets will lead to the highly localized electromagnetic field, which will then significantly enhance the Raman process in the nanojets. The main reason for obtaining different enhancements on two types of samples was analyzed.     

Surface enhanced Raman optical activity of molecules on orientationally averaged substrates: theory of electromagnetic effects

We present a model for electromagnetic enhancements in surface enhanced Raman optical activity (SEROA) spectroscopy. The model extends previous treatments of SEROA to substrates, such as metal nanoparticles in solution, that are orientationally averaged with respect to the laboratory frame. Our theoretical treatment combines analytical expressions for unenhanced Raman optical activity with molecular polarizability tensors that are dressed by the substrate's electromagnetic enhancements. We evaluate enhancements from model substrates to determine preliminary scaling laws and selection rules for SEROA. We find that dipolar substrates enhance Raman optical activity (ROA) scattering less than Raman scattering. Evanescent gradient contributions to orientationally averaged ROA scale to first or higher orders in the gradient of the incident plane-wave field. These evanescent gradient contributions may be large for substrates with quadrupolar responses to the plane-wave field gradient. Some substrates may also show a ROA contribution that depends only on the molecular electric dipole-electric dipole polarizability. These conclusions are illustrated via numerical calculations of surface enhanced Raman and ROA spectra from (R)-(-)-bromochlorofluoromethane on various model substrates.     

Characterization of hotspots in a highly enhancing SERS substrate

Vapor deposition of silver and gold onto a porous anodized aluminum oxide template is shown to produce a SERS substrate with an average surface enhancement factor of 10(7)-10(8). The high level of enhancement is explored using a combination of dark-field Rayleigh scattering and Raman spectroscopy and imaging. The scattering spectrum of the surface indicates a Plasmon resonance at 633 nm and dark-field imaging shows a relatively uniform scattering intensity at this wavelength. These measurements are consistent with the uniform enhanced Raman intensity observed in Raman maps of the substrate. Scanning electron microscopy shows the surface exhibits heterogeneous nanostructures with diameters of approximately 100 nm, the size of the pores in the template. Our measurements indicate that interactions between adjacent structures forming junctions and crevices likely give rise to a high density of hotspots, which provide the extraordinary SERS enhancement. The advantage of substrates prepared in this way is the reproducibly dense distribution of hotspots across the surface, increasing the likelihood that an analyte will experience the largest enhancement.     

Surface enhanced resonance Raman scattering of the perylene chromophore

Vibrational fundamentals, overtones and combination bands of the perylene chromophore, in the N-hexyl-3, 4:9,10-perylenetetracarboxylic diimide (HPTCNH) and other perylene tetracarboxylic derivatives, have been observed using surface enhanced resonance Raman scattering (SERRS) of Langmuir—Blodgett (LB) monolayers on Ag island films. Typical vibrational progressions due to the Franck—Condon (A-term) were seen. The results showed that the mechanism of the RRS effect was not altered by the metal surface, although the RRS signal was enhanced by four orders of magnitude. Polarization properties of the SERRS signal were studied for LB monolayers on a series of SERS active substrates. A frequency dependence of the depolarization ratios was observed.     

Near-field Raman imaging and electromagnetic field confinement in the self-assembled monolayer array of gold nanoparticles

Spatial distribution of surface enhanced Raman activity is visualized for two-dimensional (2D) nearly close-packed and well-ordered monolayer array of gold nanoparticles by using scanning near-field optical microscope. The 2D arrays exhibit highly nonuniform enhancement in Raman scattering, i.e., the regions along the edge of the 2D array are preferentially enhanced. We demonstrate that the spatial distribution of the localized electric field is also nonuniform and agrees well with that of the Raman enhancement.     

Decoration of Diatom Biosilica with Noble Metal and Semiconductor Nanoparticles (<10 nm): Assembly,Characterization, and Applications

Diatom‐templated noble metal (Ag, Pt, Au) and semiconductor (CdTe) nanoparticle arrays were synthesized by the attachment of prefabricated nanoparticles of defined size. Two different attachment techniques—layer‐by‐layer deposition and covalent linking—could successfully be applied. The synthesized arrays were shown to be useful for surface‐enhanced Raman spectroscopy (SERS) of components, for catalysis, and for improved image quality in scanning electron microscopy (SEM).     

Surface enhanced optical spectroscopies for bioanalysis

Surface enhancement can provide improved detection sensitivity in a range of optical spectroscopies. When applied to bioanalysis these enhanced techniques allow for the detection of disease biomarkers at lower levels, which has a clear patient benefit. However, to achieve widespread clinical use of surface enhanced techniques there remain several "grand challenges". In this review we consider the substrates employed to achieve enhancement before reviewing each enhanced optical technique in detail; surface plasmon resonance, localised surface plasmon resonance, surface enhanced fluorescence, surface enhanced infrared absorption spectroscopy and surface enhanced (resonance) Raman spectroscopy. Finally we set out the "grand challenges" currently facing the field.     

Chemical Enhancement and Quenching in Single-Molecule Tip-Enhanced Raman Spectroscopy

Despite intensive research in surface enhanced Raman spectroscopy (SERS), the influence mechanism of chemical effects on Raman signals remains elusive. Here, we investigate such chemical effects through tip-enhanced Raman spectroscopy (TERS) of a single planar ZnPc molecule with varying but controlled contact environments. TERS signals are found dramatically enhanced upon making a tip–molecule point contact. A combined physico-chemical mechanism is proposed to explain such an enhancement via the generation of a ground-state charge-transfer induced vertical Raman polarizability that is further enhanced by the strong vertical plasmonic field in the nanocavity. In contrast, TERS signals from ZnPc chemisorbed flatly on substrates are found strongly quenched, which is rationalized by the Raman polarizability screening effect induced by interfacial dynamic charge transfer. Our results provide deep insights into the understanding of the chemical effects in TERS/SERS enhancement and quenching.