Nanoscale structural analysis using tip-enhanced Raman spectroscopy

Tip-enhanced Raman scattering (TERS) enables the label-free investigation of biochemical interfaces with nanometer lateral resolution by combining the benefits of the intrinsic molecular specificity of Raman spectroscopy, the sensitivity because of signal enhancing capabilities of plasmonic nanoparticles, and the precision of scanning probe microscopy. The structural differentiation of constituents based on inherent molecular information is possible even down to a few nanometer spatial resolution and consequently, nucleobases, proteins, lipids, and carbohydrates can be identified and localized in a single measurement. This has been shown in the last few years for different biological samples ranging from single DNA strand investigations to cell membrane studies.     

Tip-Enhanced Raman Imaging and Nanospectroscopy: Sensitivity, Symmetry, and Selection Rules

The fundamental mechanisms of tip-enhanced Raman spectroscopy (TERS) have been investigated, including the role of the plasmonic excitation of the metallic tips, the nature of the optical tip–sample coupling, and the resulting local-field enhancement and confinement responsible for ultrahigh resolution imaging down to just several nanometers. Criteria for the distinction of near-field signature from far-field imaging artifacts are addressed. TERS results of molecules are presented. With enhancement factors as high as 10⁹, single-molecule spectroscopy is demonstrated. Spatially resolved vibrational mapping of crystalline nanostructures and determination of crystallographic orientation and domains is discussed making use of the symmetry properties of the tip scattering response and the intrinsic Raman selection rules.     

Perspectives for spatially resolved molecular spectroscopy – Raman on the nanometer scale

Nano‐objects and cellular components are of great interest in biological sciences. Tip‐enhanced Raman scattering (TERS) is a tool that allows addressing of such features for structural investigations without the need of further labelling. After brief introduction to the basic aspects of the technique an overview of present application of TERS is given. For this contribution we picked TERS experiments with emphasis on its application in life sciences. Based on these experiments, the future perspective of this analytical method and its limitations is discussed. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

利用双SLD光源提高OCT纵向分辨率的理论研究

提高光学相干层析成像(OpticalCoherenceTomography:OCT)系统纵向分辨率的关键是选取合适的光源,本文将双SLD光源代替传统OCT系统中的单个SLD光源,通过理论分析和计算机仿真,表明双SLD光源能有效提高OCT系统纵向分辨率。     

Investigation of Tip-Enhanced Raman Spectroscopy on a Silver Nanohole Array Substrate

Plasmonics - Tip-enhanced Raman spectroscopy (TERS) has received much attention due to excellent spatial resolution and high detection sensitivity. However, its performance depends crucially on the...     

Tracking of nanoscale structural variations on a single amyloid fibril with tip‐enhanced Raman scattering

Amyloid fibrils are known to be responsible for diseases such as Alzheimer's disease. A detailed insight into the structure of amyloid fibrils is fundamental since it is not yet understood what triggers the misfolding of proteins to the fiber like structures. The molecular structure of fibril surfaces on a single amino acid level has not been revealed so far but would present a valuable contribution to this question. Here we demonstrate the direct molecular distinction of selected amino acids on insulin fibril surfaces with a lateral resolution better than 2 nm by applying tip‐enhanced Raman spectroscopy (TERS). This approach provides simultaneously a way to directly reveal conformational changes in the secondary structure, namely α‐helix, β‐sheet, on the fibril surface with nanometer resolution. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Comparison and Evaluation of Silver Probe Preparation Techniques for Tip-Enhanced Raman Spectroscopy

In this work, the different procedures for the fabrication of Ag probes for tip-enhanced Raman spectroscopy (TERS) in a top illumination/detection setup are proposed and tested. We focus on technologically simple methods allowing Si tips coated with plasmonic silver nanostructures and bulk metal Ag tips with good shape reproducibility to be produced for atomic force microscopy (AFM) feedback setup. The preparation of Ag TERS probes was based on chemical deposition and vacuum sputtering of Ag on the tips of commercially available Si cantilevers. A straightforward technique for the fabrication of bulk metal Ag probes by the electrochemical etching of Ag microwires was also proposed. Chemically coated, sputtered, and electrochemically etched TERS tips were characterized by scanning electron microscopy (SEM). The produced tips were tested for TERS measurements using graphene oxide (GO) as the target analyte in a top illumination setup. A comparative analysis of enhancement factors (EF) for the different types of tips (probes) is presented in this work.     

Cover Picture: J. Biophotonics 3/2012

Tip‐enhanced Raman scattering (TERS) on amyloid fibrils – from concept to the actual experiment. A lateral resolution of two nanometers, enough to distinguish distinct amino acids on a protein, is demonstrated. Using the vibrational fingerprint of the molecules no further labeling is required and a direct identification of the primary protein structure is in reach. (Picture: T. Deckert‐Gaudig et al., pp. 215–219 in this issue)     

Selective Excitation on Tip-Enhanced Raman Spectroscopy by Pulse Shaping Femtosecond Laser

In this paper, we propose a scheme for achieving ultrafast coherent control of the selective excitation among three excited states in stimulated Raman scattering process on a tip-enhanced Raman spectroscopy (TERS). The center frequencies of the pump and Stokes laser pulses are 14,000 cm⁻¹ and 12,500 cm⁻¹, and their spectral bandwidths are both 700 cm⁻¹. By properly modulating the spectral phase distribution and cutting the frequency components, the stimulated Raman transition probabilities of two excited state keep maximal, while the other one can be suppressed to zero. The shaped pump and Stokes pulse irradiate obliquely into the TERS nanostructure containing a single layer molecule. The impulse response in temporal and frequency domain is calculated by using finite-difference time-domain (FDTD) simulation followed by Fourier transform. The frequency components and the relative phase are same with the corresponding input pulses, but the intensities are enhanced by more than 10 times. Compared with the case without the TERS nanostructure, the probability of selective excited Raman transition increases by more than 4 orders of magnitude, and the selective depressed Raman peak keeps at 0.

     

Imaging of single molecules in live cells

Progress in optical microscopy, combined to the emergence of new fluorescent probes and advanced instrumentation, now permits the imaging of single molecules in fixed and live cells. This extreme detection sensitivity has opened new modalities in cellular imaging. On the one hand, optical images with an unprecedented resolution in the 10-50 nm range, well below the diffraction limit of light, can be recorded. These super-resolution images give new insights into the properties of cellular structures. On the other hand, proteins, either in the membrane or intracellular, can be tracked in live cells and in physiological conditions. Their individual trajectories provide invaluable information on the molecular interactions that control their dynamics and their spatial organization. Single molecule imaging is rapidly becoming a unique tool to understand the biochemical and biophysical processes that determine the properties of molecular assemblies in a cellular context.     

Nanoscale distinction of membrane patches – a TERS study of Halobacterium salinarum

The structural organization of cellular membranes has an essential influence on their functionality. The membrane surfaces currently are considered to consist of various distinct patches, which play an important role in many processes, however, not all parameters such as size and distribution are fully determined. In this study, purple membrane (PM) patches isolated from Halobacterium salinarum were investigated in a first step using TERS (tip‐enhanced Raman spectroscopy). The characteristic Raman modes of the resonantly enhanced component of the purple membrane lattice, the retinal moiety of bacteriorhodopsin, were found to be suitable as PM markers. In a subsequent experiment a single Halobacterium salinarum was investigated with TERS. By means of the PM marker bands it was feasible to identify and localize PM patches on the bacterial surface. The size of these areas was determined to be a few hundred nanometers. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The structure of proteins in aqueous solutions: an assessment of triose phosphate isomerase structure by Fourier-transform infrared spectroscopy

Recent resolution enhancement and curve-fitting techniques have been applied to infrared spectra from triose phosphate isomerase in aqueous solution, in order to obtain quantitative information on its secondary structure. From our results, 57% alpha-helix, 25% beta-parallel and 10% beta-turns are predicted, in close agreement with the X-ray crystallographic data. On the other hand, measurements of band intensities, both in original and deconvolved spectra are shown to be unreliable for the quantification of secondary structures. The presence of beta-edge structure interacting with the alpha-helical barrel is described and discussed.     

Bioanalytical application of surface‐ and tip‐enhanced Raman spectroscopy

Due to its fingerprint specificity and trace‐level sensitivity, surface‐enhanced Raman spectroscopy (SERS) is an attractive tool in bioanalytics. This review reflects the research in this highly interesting topic of the last 3–4 years. The detection of the SERS signature of biomolecules up to microorganisms and cells is introduced. Labeling using modified nanoparticles (SERS tags) is also introduced. In order to establish biomedical applications, SERS analysis is performed in complex matrices such as body fluids. Furthermore, the SERS technique is combined with other methods such as microfluidic devices for online monitoring and scanning probe microscopy (i.e. tip‐enhanced Raman spectroscopy, TERS) to investigate nanoscaled features. The present review illustrates the broad application fields of SERS and TERS in bioanalytics and shows the great potential of these methods for biomedical diagnostics.     

几种超分辨率荧光显微技术的原理和近期进展

在生命科学领域,人们常常需要在细胞内精确定位特定的蛋白质以研究其位置与功能的关系．多年来,宽场/共聚焦荧光显微镜的分辨率受限于光的阿贝/瑞利极限,不能分辨出200 nm以下的结构．近年来,随着新的荧光探针和成像理论的出现,研究者开发了多种实现超出普通共聚焦显微镜分辨率的三维超分辨率成像方法．主要介绍这些方法的原理、近期进展和发展趋势．介绍了光源的点扩散函数(point spread function, PSF)的概念和传统分辨率的定义,阐述了提高xy平面分辨率的方法．通过介绍单分子荧光成像技术,引入了单分子成像定位精度的概念,介绍了基于单分子成像的超分辨率显微成像方法,包括光激活定位显微技术(photoactivated localization microscopy, PALM)和随机光学重构显微技术(stochastic optical reconstruction microscopy, STORM)．介绍了两大类通过改造光源的点扩散函数来提高成像分辨率的方法,分别是受激发射损耗显微技术(stimulated emission depletion, STED)和饱和结构照明显微技术(saturated structure illumination microscopy, SSIM)．比较了不同的z轴提取信息的方法,并阐述了这些方法与xy平面上的超分辨率显微成像技术相结合所得到的各种三维超分辨率显微成像技术的优劣．探讨了目前超分辨率显微成像的发展极限和方向．     

Electroretinographic study on the causal relationship of the S-R effect]

In the continuation of earlier studies, further serial-stimulation experiments with prolonged duration of single stimuli were carried out on dark-adapted frogs. The results have revealed a far-going dependence of the retinal response patterns (S-R effect) on the time relations between single stimuli and stimulation intervals. From all the experimental evidence available to date it can be concluded that the demonstrated serial-stimulation effects are brought about primarily in the photorecptors. According to reports in the literature these receptors in the state of dark adaptation respond to intensive, overriding stimuli with a pronounced after-effect causing a reduction of the temporal resolution capacity and of the difference susceptibility. The suppression effect can be regarded as a consequence of this overriding reaction. On the other hand, the recovery effect is caused most probably by the suddenly occurring bright adaptation because the latter makes the after-effect disappear quickly.     

Biomedical relevance of two-dimensional protein mapping

State-of-the-art and future perspectives are discussed for the application of two-dimensional protein maps to basic medical research and routine clinical chemistry problems. Despite the technical advances that allow effective processing of a large number of samples and the refinement of devices and procedures for image analysis, at present two-dimensional maps are mostly confined to research purposes, i.e. to the inventory of normal constituents of body fluids and tissues on the one hand, and to qualitative—quantitative alterations of some protein spots in a number of instances (genetic, degenerative, infectious or xenobiotic diseases) on the other. It is hoped that in some instances a single primarily affected component will be able to be identified and then specifically tested (for instance by immunological means) as a diagnostic marker, but complex pathological patterns would still require the analysis of a large number of peptides at the resolution level only afforded by two dimensions. Further simplification of the protocols, for example with ready-made gels, and data reduction systems might then allow the application of the technique to be extended to general clinical laboratories.     

Surface Characterization of Insulin Protofilaments and Fibril Polymorphs Using Tip-Enhanced Raman Spectroscopy (TERS)

Amyloid fibrils are β-sheet-rich protein aggregates that are strongly associated with a variety of neurodegenerative maladies, such as Alzheimer’s and Parkinson’s diseases. Even if the secondary structure of such fibrils is well characterized, a thorough understanding of their surface organization still remains elusive. Tip-enhanced Raman spectroscopy (TERS) is one of a few techniques that allow the direct characterization of the amino acid composition and the protein secondary structure of the amyloid fibril surface. Herein, we investigated the surfaces of two insulin fibril polymorphs with flat (flat) and left-twisted (twisted) morphology. It was found that the two differ substantially in both amino acid composition and protein secondary structure. For example, the amounts of Tyr, Pro, and His differ, as does the number of carboxyl groups on the respective surfaces, whereas the amounts of Phe and of positively charged amino and imino groups remain similar. In addition, the surface of protofilaments, the precursors of the mature flat and twisted fibrils, was investigated using TERS. The results show substantial differences with respect to the mature fibrils. A correlation of amino acid frequencies and protein secondary structures on the surface of protofilaments and on flat and twisted fibrils allowed us to propose a hypothetical mechanism for the propagation to specific fibril polymorphs. This knowledge can shed a light on the toxicity of amyloids and define the key factors responsible for fibril polymorphism. Finally, this work demonstrates the potential of TERS for the surface characterization of amyloid fibril polymorphs.     

Surface Characterization of Insulin Protofilaments and Fibril Polymorphs Using Tip-Enhanced Raman Spectroscopy (TERS)

Amyloid fibrils are β-sheet-rich protein aggregates that are strongly associated with a variety of neurodegenerative maladies, such as Alzheimer’s and Parkinson’s diseases. Even if the secondary structure of such fibrils is well characterized, a thorough understanding of their surface organization still remains elusive. Tip-enhanced Raman spectroscopy (TERS) is one of a few techniques that allow the direct characterization of the amino acid composition and the protein secondary structure of the amyloid fibril surface. Herein, we investigated the surfaces of two insulin fibril polymorphs with flat (flat) and left-twisted (twisted) morphology. It was found that the two differ substantially in both amino acid composition and protein secondary structure. For example, the amounts of Tyr, Pro, and His differ, as does the number of carboxyl groups on the respective surfaces, whereas the amounts of Phe and of positively charged amino and imino groups remain similar. In addition, the surface of protofilaments, the precursors of the mature flat and twisted fibrils, was investigated using TERS. The results show substantial differences with respect to the mature fibrils. A correlation of amino acid frequencies and protein secondary structures on the surface of protofilaments and on flat and twisted fibrils allowed us to propose a hypothetical mechanism for the propagation to specific fibril polymorphs. This knowledge can shed a light on the toxicity of amyloids and define the key factors responsible for fibril polymorphism. Finally, this work demonstrates the potential of TERS for the surface characterization of amyloid fibril polymorphs.     

Sharpening high resolution information in single particle electron cryomicroscopy

Advances in single particle electron cryomicroscopy have made possible to elucidate routinely the structure of biological specimens at subnanometer resolution. At this resolution, secondary structure elements are discernable by their signature. However, identification and interpretation of high resolution structural features are hindered by the contrast loss caused by experimental and computational factors. This contrast loss is traditionally modeled by a Gaussian decay of structure factors with a temperature factor, or B-factor. Standard restoration procedures usually sharpen the experimental maps either by applying a Gaussian function with an inverse ad hoc B-factor, or according to the amplitude decay of a reference structure. EM-BFACTOR is a program that has been designed to widely facilitate the use of the novel method for objective B-factor determination and contrast restoration introduced by Rosenthal and Henderson [Rosenthal, P.B., Henderson, R., 2003. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721-745]. The program has been developed to interact with the most common packages for single particle electron cryomicroscopy. This sharpening method has been further investigated via EM-BFACTOR, concluding that it helps to unravel the high resolution molecular features concealed in experimental density maps, thereby making them better suited for interpretation. Therefore, the method may facilitate the analysis of experimental data in high resolution single particle electron cryomicroscopy.     

Gold nanoparticle-protein arrays improve resolution for cryo-electron microscopy

Cryo-electron microscopy single particle analysis shows limited resolution due to poor alignment precision of noisy images taken under low electron exposure. Certain advantages can be obtained by assembling proteins into two-dimensional (2D) arrays since protein particles are locked into repetitive orientation, thus improving alignment precision. We present a labeling method to prepare protein 2D arrays using gold nanoparticles (NPs) interconnecting genetic tag sites on proteins. As an example, mycobacterium tuberculosis 20S proteasomes tagged with 6x-histidine were assembled into 2D arrays using 3.9-nm Au NPs functionalized with nickel-nitrilotriacetic acid. The averaged top-view images from the array particles showed higher resolution (by 6-8A) compared to analysis of single particles. The correct 7-fold symmetry was also evident by using array particles whereas it was not clear by analysis of a comparable number of single particles. The applicability of this labeling method for three-dimensional reconstruction of biological macromolecules is discussed.