Deformation history reconstruction using single quartz grain Raman microspectroscopy data

One of the most common minerals in the Earth's crust, quartz, is stable across a wide range of temperature and pressure conditions. As its microstructure is sensitive to diverse deformation mechanisms, quartz may provide valuable information regarding the structural evolution of many different rock types. Using Raman microspectroscopy, single quartz grains and monomineralic domains characterized by different deformation conditions can be identified and separated. In this study, three microstructurally extreme quartz grain types were discriminated from a subsurface shear zone: grains with undulose extinction (T0), grains with subgrains (T1), and grains with recrystallized grains (T2). Moreover, several microstructurally transitional grains were measured, which represent combinations of the aforementioned extremes. Statistical analysis revealed that the microscopically identified extreme grains possess significantly different spectral attributes and as such can be divided on the basis of certain variables of their respective Raman spectra. The three extreme quartz grain types were formed by different deformation mechanisms and thus represent distinct deformation conditions. The T0‐T1‐T2 spectral space can therefore also be considered a virtual deformational space. Although each complex quartz grain measured also appears elsewhere in the deformation process defined by T0‐T1‐T2 extreme conditions, they together represent a successive deformation path. This combined pathway is assumed to be characteristic for the whole rock volume under study. Finally, the computed Raman spectroscopy‐based virtual deformational space enabled the determination of the structural evolution of the analyzed shear zone. Copyright © 2014 John Wiley & Sons, Ltd.     

Optical sectioning using single‐plane‐illumination Raman imaging

Confocal Raman imaging is widely used for optical sectioning of materials. However, for biological applications it often suffers from poor axial resolution, photodamage to the sample of interest, substrate interference, and long acquisition times. We have applied the principles of light sheet microscopy to Raman imaging and show for the first time that optically sectioned Raman images can be obtained in significantly lower acquisition times. Copyright © 2010 John Wiley & Sons, Ltd.     

Multiplex coherent anti-Stokes Raman microspectroscopy with tailored Stokes spectrum

We combined the ultrabroadband supercontinuum of a photonic crystal fiber with a pulse shaper, resulting in a highly flexible light source for multiplex coherent anti-Stokes Raman microscopy. Implemented as the Stokes pulse, it provides tailored selection of the relevant Raman transitions, resulting in a reduced photon load and partial suppression of the nonresonant background. This experiment exploits the advantages of multiplex excitation with the increased acquisition speed of single-channel detection. The molecule-specific Stokes pulses are demonstrated for chemical mapping of a polymer blend.     

In vitro analysis of immersed human tissues by Raman microspectroscopy

Raman microspectroscopy is a powerful tool for the analysis of tissue sections, providing a molecular map of the investigated samples. Nevertheless, data pre‐processing and, particularly, the removal of the broad background to the spectra remain problematic. Indeed, the physical origin of the background has not been satisfactorily determined. Using 785 nm as source in a confocal geometry, it is demonstrated for the example of the protein kappa‐elastin that the background and resulting quality of the recorded spectrum are dependent on the morphology of the sample. Whereas a fine powder yields a dominant broad background, compressed pellets and solution‐cast thin films produce, respectively, improved quality spectra and significantly reduced spectral background. As the chemical composition of the samples is identical, the background is ascribed to stray light due to diffuse scattering rather than an intrinsic photoluminescence. The recorded spectra from a tissue sample exhibit a large and spatially variable background, resulting in poorly defined spectral features. A significant reduction of the background signal as well as improvement of the spectral quality is achieved by immersion of the sample in water and measurement with an immersion objective. The significant improvement in signal to background is attributed to a reduction of the diffuse scattering due to a change in the effective morphology as a result of an improved index matching at the water/tissue interface compared to the air/tissue interface. Compared to sections measured in air, the background is reduced to that of the water, and pre‐processing is reduced to the subtraction of the substrate and water signal and correction for the instrument response, both of which are highly reproducible. Data pre‐processing is thus greatly simplified and the results significantly more reliable. Copyright © 2010 John Wiley & Sons, Ltd.     

Phosphate and amide III mapping in sialoliths with Raman microspectroscopy

Sialoliths, a cause of the salivary gland infection, are reported to be composed of inorganic and organic substances. However, the precise mechanism of sialolith formation remains unclear. The purpose of this report is to elucidate this mechanism by analyzing the precise distribution of phosphate (an inorganic substance) and amide III (an organic substance) in sialoliths by using Raman microspectroscopy. Sialoliths from the submandibular gland duct were analyzed by this form of observation and by a scanning electron microscope (SEM) equipped with an energy‐dispersive X‐ray spectroscope (EDX). In Raman microspectroscopy we analyzed the spectral peak of the phosphate (PO₄³⁻) symmetric stretching vibrational mode (υ₁) at 960 cm⁻¹ and that of amide III at 1265 cm⁻¹ to demonstrate the mapping of an image of these elements showing a semiquantitative distribution of phosphate and amide III in the sialoliths. It was found that phosphate and amide III were concentrated at the center of the sialoliths, and the phosphate distribution in the sialoliths showed concentric laminations. These results indicated the possibility that the sialoliths originated from a nidus of organic materials and progressively grew by the deposition of layers of organic and inorganic materials. Copyright © 2008 John Wiley & Sons, Ltd.     

Functional mapping of carious enamel in human teeth with Raman microspectroscopy

We employed Raman microspectroscopy to measure the Raman spectra of phosphate in sound and carious tooth substance. The peak intensity at 960 cm⁻¹ of the phosphate (PO₄³⁻) symmetric stretching vibrational mode (υ₁) in sound enamel was stronger than that of sound dentin, which indicated that sound enamel contained more phosphate than sound dentin. Furthermore, the element analysis of phosphate in sound teeth substance, measured using a scanning electron microscope (SEM) equipped with an energy dispersive X‐ray spectroscope (EDX), gave similar results to those of the Raman measurement. In addition, the border between sound enamel and dentin was clearly demonstrated by mapping the image of the Raman spectrum of phosphate. The mapping image of phosphate in the carious enamel region revealed a heterogeneous low Raman spectrum intensity of phosphate in the area surrounding carious enamel; this finding indicates that phosphate had dissolved from the tooth substance in such areas. In contrast with the decrease in the Raman spectrum intensity of phosphate, the intensity of amide I increased mainly in the low‐phosphate area. Although it remains very difficult to clinically identify the accurate border between sound and carious tooth substance, this distinction may be enabled by using the Raman spectrum of carious tooth substance. Copyright © 2008 John Wiley & Sons, Ltd.     

Investigation of adhesive–dentin interfaces using Raman microspectroscopy and small angle X‐ray scattering

Human dentin specimens were treated with two different etch‐and‐rinse adhesives, Single Bond 2 (SB2) and Prime & Bond NT (PBNT), and two composite resins, TPH and P60. Cross‐sectional samples, approximately 1 mm thick, were analyzed with Raman line mapping and imaging across the dentin–adhesive–composite interface. The integrated intensities of selected bands associated with adhesive, organic material, composite and hydroxyapatite of dentin were calculated to determine the distribution of adhesive infiltration into demineralized dentin. The results were compared with the enamel‐adhesive composite interface. The demineralized zone was smaller in the enamel‐adhesive interface than in the dentin–adhesive interface. The region of collagen‐adhesive crosslinking was wider in the PBNT adhesive than in the SB2 adhesive. However, a gap at the dentin–PBNT composite interface, which was not observed at the dentin–SB2 composite interface, might compromise the dentin–restoration bond. K‐means cluster analysis of the Raman images confirmed the findings. The ultrastructure of the dentin–resin interface was studied using scanning electron microscopy. Small‐angle X‐ray scattering was also applied to reveal and quantify fine‐scale structural features. SB2 adhesive was found to diffuse more into demineralized dentin along with greater nanosized aggregations in the hybrid layer. Copyright © 2011 John Wiley & Sons, Ltd.     

Single-cell analysis using Fourier transform infrared microspectroscopy

Fourier transform infrared spectroscopy (FTIR) is a well-established, non-destructive method of obtaining chemical information from biological samples such as tissues and cells. This review focuses specifically on the development of infrared spectroscopic microanalysis at the single-cell level. Technological developments, including that of the infrared microscope, synchrotron radiation FTIR, and focal plane array detectors, and their impact on the field are discussed along with the various data processing procedures that are currently used to extract meaningful information. There is then an emphasis on live cell infrared (IR) imaging, including developments in water correction and microfluidic device design. The review concludes with look to future directions, highlighting the potential impact of quantum cascade lasers.     

Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells

We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.     

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.     

Confocal Raman microspectroscopy discriminates live human metastatic melanoma and skin fibroblast cells

Confocal Raman microspectroscopy (CRM) continues to develop as a promising technique with possible clinical applications for the diagnosis and treatment of skin cancers. CRM studies of single cells can provide information on the biochemical content of cancer cells in situ, potentially providing new biochemical signatures or markers of cancer cells. Here, we report a CRM study of single, living human metastatic melanoma cells (SK‐Mel‐2) and normal skin fibroblast cells (BJ) cultured and examined under identical experimental conditions. A total of almost 1200 Raman spectra were measured from more than 120 BJ and SK‐Mel‐2 cells using an inverted microscope with 647 nm laser excitation. Raman spectra were measured from within three distinct intracellular regions of the cells – cytoplasm, nucleoplasm, and nucleolus. When Raman spectra from each cell type were compared using principal components analysis (PCA) and linear discriminant analysis with leave‐one‐dish‐out cross‐validation (LDA‐CV), the two cell types were discriminated with 93% (cytoplasm), 98% (nucleolus), and 96% (nucleoplasm) accuracy. The main biochemical differences identified between the two cell types were higher RNA levels in the nucleoli of BJ cells and high amounts of lipid and collagen in the cytoplasm of SK‐Mel‐2 cells. For both cell types, higher levels of RNA were detected in the nucleoli versus the nucleoplasm. PCA with LDA‐CV was 98% (cytoplasm), 93% (nucleoplasm), and 73% (nucleolus) accurate in identifying the intracellular region based on the Raman spectra from both cell types. No significant trend was observed when the data were analyzed with respect to cell passage number. Thus, CRM with PCA and LDA‐CV successfully discriminated two skin cancer‐relevant cell lines while detecting different amounts of nucleic acids, lipids, and proteins in distinct intracellular regions, further underscoring its potential as a clinical diagnostic tool. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of organelle‐specific intracellular water structures with Raman microspectroscopy

The study of intracellular water in living cells is a challenge of fundamental importance. While the critical roles of water in maintaining and propagating life have been widely recognized and accepted, our understanding of water/biomolecule interactions is surprisingly limited. Using Raman microspectroscopic imaging of a living yeast cell followed by a multivariate analysis in form of the nonnegative matrix factorization method, we successfully resolve organelle‐specific water structures. The intensities and the band profiles of the segregated water OH stretch spectra yield important and otherwise unobtainable information on the extensive effect of the water/biomolecule interactions in a given organelle on the hydrogen‐bonding network of water molecules. Copyright © 2012 John Wiley & Sons, Ltd.     

Local observation of modes from three-dimensional woodpile photonic crystals with near-field microspectroscopy under supercontinuum illumination

A near-field microscope coupled with a near-infrared (NIR) supercontinuum source is developed and applied to characterize optical modes in a three-dimensional (3D) woodpile photonic crystal (PC) possessing a NIR partial bandgap. Spatially resolved near-field intensity distributions under different illumination wavelengths demonstrate that the electric fields preferentially dwell in the polymer rods or in the gaps between rods, respectively, for frequencies below or above the stop gap, as predicted by the 3D finite-difference time-domain modeling. Near-field microspectroscopy further reveals that the position-dependent band-edge effect plays an important role in PC-based all-optical integrated devices.     

Wide-field coherent anti-Stokes Raman scattering microscopy with non-phase-matching illumination

We have developed and tested a wide-field coherent anti-Stokes Raman scattering (CARS) microscopy technique, which provides the simultaneous imaging of an extended illuminated area without scanning. This method is based on the non-phase-matching illumination of a sample and imaging of a CARS signal with a CCD camera using conventional microscope optics. We have identified a set of conditions on the illumination and imaging optics, as well as on sample preparation. Imaging of test objects proved high spatial resolution and chemical selectivity of this technique.     

胃粘膜组织共焦显微拉曼光谱分类研究

采用基于小波变换的光谱去噪和背景扣除预处理技术,对32例(其中:13例正常,19例癌变)胃粘膜组织拉曼光谱进行分析,克服了手动背景扣除的一些缺点,并观察到一些新的光谱特征。基于此,给出能对所有正常组织和癌变组织进行有效分类的特征量和判据。通过研究这些特征量与胃粘膜组织癌变发展阶段之间的定量关系,可望实现胃癌的早期诊断。     

How to obtain a reliable structural characterization of polished graphitized carbons by Raman microspectroscopy

A series of graphitized carbon materials, produced by the pyrolysis of an anthracene‐based coke at temperatures ranging from 1600 to 2900 °C, were studied by Raman microspectroscopy to assess the applicability of this technique to the particular case of polished carbon materials. The polishing process was shown to change significantly the first‐order Raman spectra (D band intensity increase) and therefore to induce unacceptable errors in the characterization of the intrinsic structure of these materials. The deconvolution of Raman spectra, related to the unpolished graphitized carbons at varying temperatures, highlighted a linear relationship between the intensity ratio I_D/I_G and the G band width. Thus, as the latter appears to be insensitive to the polishing, we highly recommend using it for a reliable assessment of the intrinsic structural disorder of polished carbon materials. Copyright © 2011 John Wiley & Sons, Ltd.     

High concentration trans form unsaturated lipids detected in a HeLa cell by Raman microspectroscopy

High concentration trans form unsaturated lipids have been found in a HeLa cell by Raman microspectroscopy. Two CC stretch bands are observed simultaneously at 1669 cm⁻¹ (trans form) and at 1656 cm⁻¹ (cis form) in a Raman spectrum obtained from a small area (1 µm in diameter) in a HeLa cell. The intensity ratio 1669/1656 indicates that the concentration of the trans form is as high as that of the cis. It is demonstrated that Raman microspectroscopy provides a powerful and unique means for in situ and noninvasive structural characterization of unsaturated lipids in a living cell. Copyright © 2008 John Wiley & Sons, Ltd.