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.     

Micro‐Raman mapping on polished cross sections: a tool to define the penetration depth of conservation treatment on cultural heritage

Nowadays, in order to preserve the surfaces of works of art, several organic and inorganic substances are used. To evaluate the penetration depth and the distribution of these substances inside the materials, it is essential to define the effectiveness of a conservative treatment. Owing to the ambiguous results obtained with the current analytical techniques, the achievement of this aim is critical. This study shows how micro‐Raman mapping on polished cross section is an effective tool capable of investigating the inner portions of the materials. A set of painted plasters has been treated with ammonium oxalate solutions, an inorganic substance widely used for conservation works of carbonatic materials. The micro‐Raman mapping of the lines of the newly formed calcium oxalate allowed correlation of the penetration depth and the distribution of the product inside the specimens with different conditions of treatment. Moreover, a good agreement between micro‐Raman spectroscopy and microscopic techniques has been obtained. Copyright © 2010 John Wiley & Sons, Ltd.     

Micro‐Raman depth profiling on polished cross‐sections: the mapping of oxalates used in protective treatment of carbonatic substrate

We present the first depth profiling on a polished cross‐section carried out by micro‐Raman spectroscopy aimed at investigating the penetration depth of an inorganic protective material (ammonium oxalate) applied on carbonatic surfaces (ancient plaster) and to detect the distribution of mineralogical phases inside the material. The reason for such a Rapid Communication is the importance of our study for the field of conservation, in which at present the treatment with oxalates is a hot issue. Copyright © 2008 John Wiley & Sons, Ltd.     

Passive signal enhancement in spatially offset Raman spectroscopy

We demonstrate experimentally, for the first time, the feasibility of enhancing signals in Spatially Offset Raman Spectroscopy (SORS) using a dielectric bandpass filter, building on our earlier experimental work on the enhancement of transmission Raman signals. The method is shown to lead to the enhancement of both the surface and subsurface Raman layer signal improving the signal‐to‐noise ratio of Raman spectra from the deep areas of samples, thus enhancing the technique's sensitivity and penetration depth. The filter is placed over the laser illumination zone, on the sample surface acting as a ‘unidirectional’ mirror transmitting the collimated laser beam on one side and reflecting photons escaping from the sample back into it. This enhances the degree of coupling of laser radiation into the medium and associated generated Raman signal. The feasibility study was performed on a two‐layer sample with the second layer located at the limit of the penetration depth of the method for this sample. The sample consisted of a 2.2‐mm over‐layer of a thinned paracetamol tablet followed by a 2‐mm layer of trans‐stilbene powder. The Raman signal was collected from a spatially offset region through a hole fabricated within the filter. The experiments demonstrate the presence of an enhancement of the Raman signal from both the layers by a factor of 4.4–4.5 and the improved signal‐to‐noise ratio of sublayer signal by a factor of 2.2, in agreement with photon shot noise dominated signal. Copyright © 2008 John Wiley & Sons, Ltd.     

基于共聚焦显微拉曼的真菌菌丝中几丁质的原位检测研究

几丁质是真菌细胞壁中一种重要的结构多糖,本文首次采用共聚焦显微拉曼技术对山茶刺盘孢菌的气生菌丝进行原位检测研究,首先确定了采集菌丝拉曼光谱的最优实验参数,并获得了菌丝,几丁质标准品和背景三种物质的典型拉曼光谱,对其中的特征峰进行归属分析,发现菌丝光谱中有明显的几丁质特征峰。然后对置于载玻片上菌丝的感兴趣区域进行拉曼光谱面扫描,通过主成分分析法发现面扫描区域中,菌丝和背景两种信号可以明显区分开来,结合主成分的载荷因子图得到了菌丝的两个主要的特征差异峰1 622和1 368 cm-1,1 622 cm-1属于菌丝中几丁质的特征峰,而1 368 cm-1是来源于菌丝中的果胶多糖。最后通过对几丁质在1 622 cm-1特征峰波段附近范围积分,绘制了几丁质在菌丝中二维和三维的化学成像图,直观且无损的再现了几丁质在菌丝中的空间分布。     

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.     

Non‐invasive depth profiling by space and time‐resolved Raman spectroscopy

Time‐resolved Raman spectroscopy, spatially offset Raman spectroscopy and time‐resolved spatially offset Raman spectroscopy (TR‐SORS) have proven their capability for the non‐invasive profiling of deep layers of a sample. Recent studies have indicated that TR‐SORS exhibits an enhanced selectivity toward the deep layers of a sample. However, the enhanced depth profiling efficiency of TR‐SORS, in comparison with time‐resolved Raman spectroscopy and spatially offset Raman spectroscopy, is yet to be assessed and explained in accordance to the synergistic effects of spatial and temporal resolutions. This study provides a critical investigation of the depth profiling efficiency of the three deep Raman techniques. The study compares the efficiency of the various deep Raman spectroscopy techniques for the stand‐off detection of explosive precursors hidden in highly fluorescing packaging. The study explains for the first time the synergistic effects of spatial and temporal resolutions in the deep Raman techniques and their impact on the acquired spectral data. Copyright © 2013 John Wiley & Sons, Ltd.     

Confocal Raman spectroscopy to monitor intracellular penetration of TiO2 nanoparticles

Confocal Raman microscopy, a noninvasive, label‐free, and high‐spatial resolution imaging technique, in combination with K‐mean cluster analysis and a correlation coefficient map, was employed to trace titanium dioxide (TiO₂) nanoparticles in living MCF‐7 and TERT cells. The penetration of TiO₂ nanoparticles into cells revealed a gradual time‐dependent diffusion of nanoparticles over the entire cell. Cell apoptosis was monitored by tracing cytochrome c diffusion into the cytoplasm. A comparison with the mitochondrial clustering indicated that cytochrome c was inside the mitochondria for TiO₂ concentration of 2 µg ml⁻¹. This result demonstrates that the presence of TiO₂ particles within a cell does not induce apoptosis. We demonstrated that confocal Raman microscopy allow to follow penetration of TiO₂ particles in cell and to monitor the apoptotic status of the penetrated cells. Copyright © 2014 John Wiley & Sons, Ltd.     

拉曼高光谱成像系统中光在奶粉层的穿透深度研究

拉曼高光谱成像技术不仅可以获取样本的空间分布信息,图像上每个像素点还包括了完整的光谱信息,因其信息量丰富的特点已在食品安全检测方面得到了应用。本研究探索拉曼高光谱成像系统中光在奶粉层中的穿透深度,以及采集参数和奶粉类型对穿透深度的影响。实验选取均匀奶粉层样品放置于厚度为5 mm的三聚氰胺样本之上,检测奶粉层厚度为0.8～4.0 mm时的三聚氰胺特征峰强度,以此评估光在奶粉层中的穿透性和信号衰减情况。结果显示当奶粉层厚度一定时,随着激光功率变大,拉曼特征峰值随之增加,此外更长的曝光时间也可以使拉曼信号得到增强。在激光功率不小于2 W且曝光时间不小于500 ms时,光在全脂奶粉层的穿透深度可达4 mm。奶粉层厚度在0.8～4.0 mm范围内,穿透奶粉层的拉曼信号随着奶粉层厚度增加呈指数式衰减。在激光功率为8 W、曝光时间为1 000 ms的条件下,光在全脂、低脂和脱脂奶粉层的穿透深度均达到了4 mm。在相同测量厚度下,通过脱脂奶粉层接收的拉曼信号弱于通过全脂和低脂奶粉层接收的拉曼信号强度。研究结果为拉曼高光谱检测中奶粉样品的前处理提供了有益参考。     

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.     

Raman scattering investigation of selenomethionine replacement in protein SOUL crystals

The vibrational properties of both wild‐type and selenomethionine (SeMet)‐substituted protein SOUL crystals have been investigated here by Raman spectroscopy. Several Raman peaks observed in the spectra of methionine and SeMet were identified as specific markers. The unambiguous assignment of these peaks has been inferred by comparing the experimental Raman spectra of the pure amino acids, recorded in solid state and in aqueous solution, and the Raman intensities computed using quantum chemical calculations. Moreover, a quantitative evaluation of the relative amount of SeMet replacement in the crystals of protein SOUL labelled with SeMet has been estimated through the ratio between the Raman intensities of marker peaks. These results offer evidence of the potential of Raman microscopy as a reliable and non‐invasive tool for novel in‐depth structural investigations in biocrystallography. Copyright © 2009 John Wiley & Sons, Ltd.     

Detection of explosives on human nail using confocal Raman microscopy

Trace amounts of explosives were detected on human nail using confocal Raman microscopy. Contamination of the nail can result from the manual handling, packaging or transportation of explosive substances. Raman spectra were obtained from pentaerythritol tetranitrate (PETN), trinitrotoluene (TNT), ammonium nitrate and hexamethylenetetramine (HMTA) particles on the surface of the nail with dimensions in the range 5–10 µm. An added difficulty in an analytical procedure is the presence of a nail varnish coating that has been applied, which traps the particulate matter between the coating and nail. Using confocal Raman microscopy, interference‐free spectra could be acquired from particles of explosives visually masked by the nail varnish. Spectra of the explosives could be readily obtained in situ within 90 s without alteration of the evidential material. Acquisition of a Raman point map of a PETN particle under the nail varnish coating is also reported. Copyright © 2008 John Wiley & Sons, Ltd.     

Fast mapping of inhomogeneities in the popular metallic perovskite Nb:SrTiO3 by confocal Raman microscopy

Confocal Raman microscopy was applied in order to investigate the homogeneity of donor doping in Nb:SrTiO₃ single crystals. Measurements of local Raman spectra revealed a systematic relation between the intensity of the Raman signal and the donor content of the crystals. We successfully elaborated a correspondence between the electronic structure and the intensity of the Raman lines using a crystal with macroscopic inhomogeneity as a demonstration sample. By mapping the distribution of the intensity of the Raman signal, we identified a characteristic inhomogeneous structure related to the presence of clusters with sizes of 5 µm to 20 µm, indicating inhomogeneous donor distribution caused by flaws introduced during crystal growth. Hence, we propose confocal Raman microscopy as a convenient technique for investigating the homogeneity and quality of doped perovskite surfaces, which are needed for various technological applications. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Raman spectroscopy and microscopy of individual cells and cellular components

Raman spectroscopy provides the unique opportunity to nondestructively analyze chemical concentrations in individual cells on the submicrometer length scale without the need for optical labels. This enables the rapid assessment of cellular biochemistry inside living cells, and it allows for their continued analysis. Here, we review recent developments in the analysis of single cells, subcellular compartments, and chemical imaging based on Raman spectroscopy. Spontaneous Raman spectroscopy provides for the full spectral assessment of cellular biochemistry, while coherent Raman techniques, such as coherent anti‐Stokes Raman scattering is primarily used as an imaging tool comparable to confocal fluorescence microscopy. These techniques are complemented by surface‐enhanced Raman spectroscopy, which provides higher sensitivity and local specificity, and also extends the techniques to chemical indicators, i.e. pH sensing. We review the strengths and weaknesses of each technique, demonstrate some of their applications and discuss their potential for future research in cell biology and biomedicine.     

基于最大似然法的共焦拉曼光谱成像方法

随着现代科技对纳米微观区域兴趣的增加,如DNA测序、分子纳米器件微结构检测等,其对拉曼光谱技术的空间分辨力提出了更高的要求,而现有共焦拉曼光谱技术受自身原理限制,空间分辨力已无法满足科学需求。针对这一问题,在现有共焦拉曼光谱技术的基础上,提出一种基于最大似然算法的共焦拉曼光谱成像方法。该方法将超分辨图像复原技术与共焦拉曼光谱技术相结合,利用基于Poisson-Markov约束的最大似然超分辨复原算法对共焦拉曼光谱图像进行超分辨图像复原处理,恢复图像高频成分,进而改善共焦拉曼光谱系统的空间分辨能力,实现超分辨成像。仿真分析和实验结果表明,提出的基于最大似然算法的共焦拉曼光谱成像方法在不改变现有共焦拉曼光谱系统光学结构的前提下,仅对单幅拉曼光谱图像进行超分辨图像复原处理,即可将系统空间分辨力提高到200 nm,实现超分辨成像,同时该方法具有较强的噪声抑制能力。该方法有效地提高了共焦拉曼光谱系统的空间分辨力,为物理化学、材料科学等前沿领域中的高空间分辨微区光谱探测提供了一种新的途径,是一种行之有效的高空间分辨的共焦拉曼光谱成像方法。     

Characterization of tetragonal SAT0.3: LA0.075: CAT0.625 perovskite crystal: spectroscopic and microscopic investigations

We report room temperature measurements of X‐ray diffraction (XRD), optical transmission microscopy (OTM), atomic force microscopy (AFM), infrared‐absorption (IR), and micro‐Raman spectroscopy (µ‐RS) of the oriented SAT_0.3: LA_0.075: CAT_0.625 single crystal. The final structure refinement of SAT_0.3: LA_0.075: CAT_0.625 crystal was performed for I4/m space group at room temperature. Vibrational spectra of the crystal were discussed in terms of group‐theoretical predictions for untilted (Fm3 m) and tilted tetragonal (I4/m) perovskite structure. The confocal µ‐Raman measurements of depth profiling of SAT_0.3: LA_0.075: CAT_0.625 crystal suggest a relationship between sensitivities of the ordering‐related Raman‐active modes and the variation of order parameter η. Copyright © 2009 John Wiley & Sons, Ltd.     

人全血的毛细管显微拉曼光谱分析

血液中含有众多生物信息,如激素、酶、抗体等丰富的蛋白质成分。通过对血液中众多生物信息进行检测鉴定可以起到对该血液种属判定、溯源的目的。因此,血液检测技术的发展在诸如刑事案件侦破、物种鉴定、疾症预防等领域具有重要意义。目前,传统血液检测手段多为显微观测、免疫法、DNA/基因检测法等,这些技术会对血液样本造成不可逆转的破坏性,且存在分析周期长、结构装置复杂、试验价格昂贵等问题。随着激光技术的发展,拉曼光谱技术作为一种非线性散射光谱技术,在血液检测技术中得到了应用。在血液检测技术中,拉曼光谱技术通常与共聚焦显微系统结合,对涂在载玻片上或盛放在透明容器中的血液样品进行光谱信号采集。该技术具有快速、无损等优势,但复杂的光路系统及昂贵的实验装置限制了该技术的广泛推广。为提出一种装置简单、操作简便的血液拉曼检测新技术,研究采用基于毛细管的显微拉曼技术方案采集并分析人全血的拉曼信号。血液样品通过毛细管的虹吸效应取样,与载玻片的涂样方式相比毛细管的方案具有模拟人血管、维持血液活性、减小空气对实验过程中血液成分的影响、降低激光对血液样品的灼伤效果等优势。为避开可见光部分荧光较强区域的荧光干扰,研究采用360 nm紫外激光器作为激发光源,防止可见荧光信号的干扰。积分时间设为800 ms,有效避免因激光长时间照射对血液样品的灼伤效果,影响实验数据的稳定性与真实性,光谱平均次数为2次,避免单次测量所带来的数据的不准确性影响。光谱扫描范围为500～1 800 cm-1, 结果表明此范围内可较好的避开可见光部分荧光较强区域的干扰。测得的拉曼光谱信号通过滤波去噪及基线校正进行处理。首先采用5阶离散小波变换滤波,进行1层信号分解,滤除高频噪声信号,保留低频有效信号,从而去除杂散信号,对光谱有效信号进行提取。其次,采用4阶多项式拟合扣除基底的基线校正,实现人全血的毛细管显微拉曼光谱峰值信号的提取。最终,通过查询SDBS数据库以及人血样本通过reishaw共聚焦显微拉曼光谱仪测量所得光谱图进行验证发现测得信号中部分为人体内数种氨基酸成分的拉曼信号。实验研究发现,基于毛细管的显微拉曼实验系统与常规拉曼探头实验系统相比,拉曼信号更稳定、重复性高,可有效提取人全血中的拉曼光谱信号, 而其与高精度的共聚焦显微拉曼系统相比价格便宜、结构简单、易于推广等优点,但信号信噪比、有效信号的峰值强度上仍有进一步的提升,是一种测量人全血拉曼信号的可行方案。     

内分泌干扰物三苯基锡的激光拉曼光谱分析

三苯基锡(TPhT)是目前已知的内分泌干扰物中唯一的两种金属化合物之一,被广泛应用于工业、农业和交通领域,其大量使用会对土壤、海洋和内陆淡水环境造成不同程度的影响。本实验采用激光共聚焦拉曼光谱采集固体TPhT的拉曼光谱信号,尝试将该方法用于TPhT检测,探索该方法的可行性,并进行检测参数的优化选择。将拉曼光谱分析检测方法与TPhT的物性研究相结合,根据TPhT分子中不同官能团振动模式的不同,将拉曼谱图分为高、中、低3个波数区(1 500～3 200,900～1 500和100～900 cm-1)进行拉曼谱峰的归属与分析,得到了TPhT的特征振动模式和拉曼特征峰,并建立一套TPhT的标准拉曼图谱库,光谱范围在100～3 200 cm-1之间。结果表明,当激光功率选择为衰减到原激光功率(500 mW)的0.5%、曝光10 s、累积2次时,得到的拉曼谱图信噪比高且检测时间短。在212,332,657,997和1 577 cm-1处出现的信号强度较高的拉曼峰,可作为固体TPhT拉曼检测的特征峰,657和997 cm-1处拉曼特征峰的同时出现即可认为复杂的环境样品中存在TPhT。实验结果给出了辨别TPhT存在的标志,这些结果将为拉曼光谱用于实际环境样品中TPhT的残留检测提供理论依据和数据基础。     

Comparison of molecular images as defined by Raman spectra between normal mucosa and squamous cell carcinoma in the oral cavity

Raman spectroscopy has been effectively applied to clinically differentiate normal and cancerous mucosal tissues. Micro‐Raman spectroscopy provides a tool to better understand the molecular basis for the Raman clinical signal. The objective of the current study was to utilize micro‐Raman spectroscopy to define the molecular/spectral differences between normal and abnormal squamous cell carcinoma (SCC) in oral mucosa (in vitro). Understanding this may help in identifying unique spectra or may be useful for in vivo application of this technology. Micro‐Raman (confocal) spectroscopy was used to obtain molecular images of normal and SCC cells of human oral mucosa. Four fresh flashed‐frozen tumor and four matched normal tongue specimens were studied. The spectra covered a wavenumber range from 300 to 4000 cm⁻¹ with a spectral resolution of 8 cm⁻¹ and a spatial resolution of 1.0 µm. The cells were located within thin sections of tongue mucosa biopsies. The excitation wavelength of 515 nm was used. We were able to obtain Raman images with rich information about the spectroscopic and structural features within the cytoplasm, cell membrane, and cell nuclei. Significant spectral differences were observed between the Raman images of normal and malignant squamous cells. The heterogeneity of tumor cells within the abnormal tissue was also demonstrated. Spectral differences demonstrated between both tissue types have provided important information regarding the origins of specific signals within the cells of each tissue type. In our search for specific spectral biomarkers, we believe that a cell surface protein, greatly upregulated in SCC cells, was discovered at 1583 cm⁻¹. Copyright © 2010 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman microscopy of hemispherical shells stripped from templates of anodized aluminum

We report on the optical characterization of plasmonic metal nanostructures representing highly ordered interconnected hemispherical gold and silver shells that can be iteratively stripped from the same embossed templates (without template degradation) made from selectively etched anodized aluminum. By performing scanning high‐resolution confocal Raman microscopy of p‐aminothiophenol and Rhodamine 6G molecules homogeneously adsorbed to samples with different radii of shell curvature, we systematically investigate the applicability of the fabricated structures for surface‐enhanced Raman spectroscopy and correlate the results with linear reflection spectroscopy. We trace the origin of strong Raman signal enhancements (average relative enhancement of up to ~120) to electromagnetic hot‐spots located in sharp grooves and crevices at hemisphere shell junctions. Copyright © 2011 John Wiley & Sons, Ltd.