Characterization of pediatric Wilms' tumor using Raman and fluorescence spectroscopies

Purpose

Raman spectroscopy has been successfully demonstrated as an effective tool for tissue characterization and diagnosis, but nearly all studies have interrogated adult tissues and diseases. In this study, we demonstrate the application of Raman spectroscopy and its background autofluorescence for pediatric Wilms' tumor diagnosis.

Methods

Eight tumors were measured in this study, along with matched normal kidney tissue in 6 cases. Spectral comparisons were drawn, and diagnostic use was assessed using both the Raman spectral features as well as the inherent tissue fluorescence.

Results

The fluorescent background spectra were able to discriminate normal kidney from Wilms' tumor with 81% sensitivity and 100% specificity. The Raman spectra obtained 93% sensitivity and 100% specificity.

Conclusions

This pilot study shows that both autofluorescence and Raman spectra provide diagnostic use in discriminating Wilms' tumor from normal kidney. These techniques may be used individually or in tandem to develop a real-time intraoperative screening and diagnostic device.     

高灵敏性表面增强拉曼散射传感器的构建及其用于人体尿液中痕量甲醛的快速检测

目的：构建响应快速、灵敏度高的表面增强拉曼散射(surface-enhanced Raman scattering,SERS)传感器,实现对人体尿液中痕量甲醛的快速检测。方法：以对-氨基苯硫酚(p-ATP)为探针分子,通过表面自组装技术在银纳米粒子表面修饰具有SERS信号响应的单分子层,滴加甲醛后,甲醛通过醛基与p-ATP发生特异性缩合反应,p-ATP原有拉曼信号强度猝灭,进而实现对甲醛的间接检测。结果：甲醛在1.0×10-8~1.0×10-7 mol/L范围内呈现良好的线性,相关系数为0.991 7,检测限为6.5×10-9 mol/L,回收率为97.5%~106.0%。结论：本方法可用于人体尿液中痕量甲醛的检测,具有耗时少、简便和灵敏度高等优点。     

Interfacial hydration determines orientational and functional dimorphism of sterol-derived Raman tags in lipid-coated nanoparticles

Lipid-coated noble metal nanoparticles (L-NPs) combine the biomimetic surface properties of a self-assembled lipid membrane with the plasmonic properties of a nanoparticle (NP) core. In this work, we investigate derivatives of cholesterol, which can be found in high concentrations in biological membranes, and other terpenoids, as tunable, synthetic platforms to functionalize L-NPs. Side chains of different length and polarity, with a terminal alkyne group as Raman label, are introduced into cholesterol and betulin frameworks. The synthesized tags are shown to coexist in two conformations in the lipid layer of the L-NPs, identified as “head-out” and “head-in” orientations, whose relative ratio is determined by their interactions with the lipid–water hydrogen-bonding network. The orientational dimorphism of the tags introduces orthogonal functionalities into the NP surface for selective targeting and plasmon-enhanced Raman sensing, which is utilized for the identification and Raman imaging of epidermal growth factor receptor–overexpressing cancer cells.

Lipid-coated noble metal nanoparticles (L-NPs) that contain a lipid membrane around a NP core represent an intriguing class of hybrid materials that combine the surface properties of biological membranes with the advantageous material properties and optical responses of a metal NP core (1–5). Depending on their exact composition, lipid membranes can enable the conjugation of specific biomolecules (6), facilitate the mimicry of biological NPs such as exosomes or enveloped viruses (7), engage cellular lipid-binding functionalities and associated signaling pathways (7–9), and provide custom-tailored stealth properties for biomedical applications (10). The noble metal NP core sustains strong size and shape-dependent localized surface plasmon resonances (LSPRs), which give rise to large, optical absorption and scattering cross-sections. The strong electric field (E-field) within the vicinity of the NPs under resonant excitation of the LSPR can enhance the inelastic scattering of molecules localized in the evanescent E-field close to the metal surface. This well-known effect forms the foundation of surface enhanced Raman spectroscopy (SERS) (11, 12). The large electron and X-ray absorption cross-sections also render noble metal NPs versatile and multimodal probes for optical, electron, and X-ray microscopies (13, 14).Due to their structural similarity with enveloped viruses, L-NPs have been used as model systems for investigating the viral binding and entrance (5, 15), as well as for probing intracellular trafficking pathways (7–9). L-NPs have also been exploited for high-contrast biomolecular imaging (16, 17), for controlled drug or cargo delivery (18, 19), and for plasmon-enhanced pathogen inactivation (20, 21). Importantly, the functionality of L-NPs in specific applications can be determined by the structure and composition of the lipid architectures. For instance, L-NPs intended for targeting cell surface receptors in particle–cell interaction studies (8, 22) or for enhancing photochemical reactions in the ambient medium (4) require targeting ligands or photocatalysts localized in the membrane surface, where they are accessible to the medium. In contrast, optical probe (16, 23) or nanocarrier (18, 21) applications of L-NPs benefit from an integration of optical labels or cargo molecules within the membrane, where they are protected from environmental influences and reactions.Sterols, in particular cholesterol, and other terpenoid derivatives are important components of L-NPs and, in general, play vital roles in regulating membrane formation (24–26), fluidity (27), phase behavior (28, 29), domain formation (30, 31), permeability (32), and additional physicochemical properties of both biological and artificial lipid architectures. Therefore, chemical derivatization of these essential membrane components presents a promising strategy for controlling the membrane properties of L-NPs. We modify, in this work, cholesterol and betulin to generate three modified tag molecules (Tag-1 to Tag-3) with side chains that differ in polarity and lengths, but all contain a terminal alkyne group, and investigate their integration into L-NPs. The tag structure as well as the solvation properties of the modified side chains impact their interactions with surrounding lipids and water, determine their orientation in the membrane, and thus directly affect the surface chemical properties of the L-NPs. The terminal alkyne provides a platform for bioorthogonal chemical conjugation, as well as a strong and unique Raman signature whose precise stretching frequencies depend on the local chemical environment (33, 34). We probe the orientations of Tag-1 to Tag-3 in the NP-supported lipid membrane, elucidate the underlying mechanisms that determine the orientation of the tags in the membrane, and utilize them to engineer plasmon-enhanced Raman probes for the detection of epidermal growth factor receptor (EGFR)-overexpressing cancer cells.     

拉曼光谱法测定硫普罗宁注射液的含量

 目的 建立拉曼光谱法测定硫普罗宁注射液含量的方法。方法 利用拉曼散射原理,在水溶液中,拉曼峰信号的强度与溶质分子的浓度成正比,选取样品溶液拉曼光谱中硫普罗宁的巯基S-H振动(2 580 cm^-1)处的强峰作为定量峰,建立拉曼光谱法测定硫普罗宁注射液含量的方法。结果 硫普罗宁在10～100 mg·mL^-1内与拉曼峰强度呈良好的线性关系,线性回归方程为Y=166.1ρ-30.92,线性相关系数为0.999 9（n=6）,平均回收率为101.98%,RSD为2.09%（n=9）。结论 本方法操作简便、快速,可用于硫普罗宁注射液的含量测定。     

拉曼光谱法快速测定盐酸异丙嗪注射液的含量

目的:建立拉曼光谱法快速测定盐酸异丙嗪注射液的含量。方法:采用智能激光拉曼光谱仪扫描样品,激发波长780 nm,激光强度100 MW,光圈25 μm,光栅400 gr·mm^-1,分辨率2.0 cm^-1,曝光时间50 s,曝光次数2次,扫描范围100~3 200 cm^-1。结果:盐酸异丙嗪在20~200 mg·ml^-1范围内与拉曼定量峰面积呈良好的线性关系(r=0.998 2),平均加标回收率为98.13%,RSD为1.05%(n=9)。结论:本方法快速、简单、可靠,可用于盐酸异丙嗪注射液的含量测定。     

Label‐free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis

The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti‐Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two‐photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG‐active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co‐localized with positive albumin immunohistochemistry and might indicate degeneration‐associated vascular leakage. We present a label‐free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label‐free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo.     

Raman spectroscopy for early real-time endoscopic optical diagnosis based on biochemical changes during the carcinogenesis of Barrett’s esophagus

Raman spectroscopy is a spectroscopic technique based on the inelastic scattering of monochromatic light that represents the molecular composition of the interrogated volume to provide a direct molecular fingerprint. Several investigations have revealed that confocal Raman spectroscopy can differentiate non-dysplastic Barrett’s esophagus from esophageal high-grade dysplasia and adenocarcinoma with high sensitivity and specificity. An automated on-line Raman spectral diagnostic system has made it possible to use Raman spectroscopy to guide accurate target biopsy instead of multiple random forceps-biopsies,this novel system is expected to improve in vivo precancerous diagnosis and tissue characterization of Barrett’s esophagus.     

Freeze‐drying as a preserving preparation technique for in vitro testing of human skin

In vitro testing of drugs with excised human skin is a valuable prerequisite for clinical studies. However, the analysis of excised human skin presents several obstacles. Ongoing drug diffusion, microbial growth and changes in hydration state influence the results of drug penetration studies. In this work, we evaluate freeze‐drying as a preserving preparation method for skin samples to overcome these obstacles. We analyse excised human skin before and after freeze‐drying and compare these results with human skin in vivo. Based on comprehensive thermal and spectroscopic analysis, we demonstrate comparability to in vivo conditions and exclude significant changes within the skin samples due to freeze‐drying. Furthermore, we show that freeze‐drying after skin incubation with drugs prevents growth of drug crystals on the skin surface due to drying effects. In conclusion, we introduce freeze‐drying as a preserving preparation technique for in vitro testing of human skin.     

矿物药雄黄的红外及拉曼光谱鉴定

目的:对3批雄黄生品及1批雄黄饮片进行红外光谱、拉曼光谱研究.方法:利用傅立叶红外光谱法、激光拉曼光谱并结合二阶导数拉曼光谱对各雄黄样品进行鉴别分析.结果:各样品的红外光谱在3 400,1 630 cm-1附近出现了吸收峰,在1 500～400 cm-1也出现了吸收峰,但均不是雄黄的红外特征吸收峰,各样品的拉曼光谱及其二阶导数谱均在182,234,271,342 cm-1等波数位置出现了特征性较强的拉曼振动峰.结论:红外光谱法不适用于雄黄的特征鉴别,拉曼光谱能较好地反映出雄黄的特征吸收峰,具有快速、简便、准确等优点,该法值得在中药鉴定中得到更广泛的应用.     

SERS and resonance Raman studies of p-aminoazobenzene on gold and silver electrodes

Surface-enhanced Raman scattering (SERS) spectra and cyclic voltammetry were used to detect the products of the oxidation and reduction of p-aminoazobenzene (PAAB) on roughened gold and silver electrodes in acidic and neutral solutions.The results indicated that oligomerization products are formed during the oxidation of PAAB on a gold electrode. We identified these products as oligomers formed by CN coupling of the PAAB cation radicals. The same products are formed on a silver electrode at a lower potential as the result of a photoelectrochemical reaction.The measurements carried out in the negative potential range proved that PAAB undergoes irreversible reduction in acidic media and reversible reduction to hydrazobenzene in neutral media.