Raman spectroscopy analysis of lipid droplets content,distribution and saturation level in Non‐Alcoholic Fatty Liver Disease in mice

Non‐Alcoholic Fatty Liver Disease (NAFLD) is a common liver disorder, characterized by an excessive lipids deposition within the hepatic tissue. Due to the lack of clear‐cut symptoms and optimal diagnostic method, the actual prevalence of NAFLD and its pathogenesis remains unclear, especially in the early stages of progression. In the presented work confocal Raman microspectroscopy was used to investigate alterations in the chemical composition of the NAFLD‐affected liver. We have investigated two NAFLD models, representative for macrovesicular and microvesicular steatosis, induced by High Fat Diet (60 kcal %) and Low Carbohydrate High Protein Diet (LCHP), respectively. In both models we confirmed the development of NAFLD, manifested by the presence of lipid droplets (LDs), but of different sizes. Model of macrovesicular steatosis was characterized by large LDs, whereas in the microvesicular steatosis model small droplets were found. In both models, however, we observed a significant decrease in the degree of unsaturation of lipids, in comparison to the control. In addition, for both models, the impact of medical treatment with selected drugs (perindopril and nicotinic acid, respectively) was tested, indicating a significant influence of medicine not only on the occurrence and size of the droplets, but also on their composition. Inboth cases the drug treatment resulted in an increase of the degree of unsaturation of lipids forming droplets. Confocal Raman microspectroscopy was proven to be a powerful tool providing detailed insight into selected areas of hepatic tissue, following the NAFLD pathogenesis and diagnostic potential of the applied drugs.

     

At-line raman spectroscopy and design of experiments for robust monitoring and control of miniature bioreactor cultures

The biopharmaceutical industry is moving toward a more quality by design (QbD) approach that seeks to increase product and process understanding and process control. Miniature bioreactor systems offer a high-throughput method enabling the assessment of numerous process variables in a controlled environment. However, the number of off/at-line samples that can be taken is restricted due to the small working volume of each vessel. This limitation may be resolved through the use of Raman spectroscopy due to its ability to obtain multianalyte data from small sample volumes fast. It can, however, be challenging to implement this technique for this application due to the complexity of the sample matrix and that analytes are often present in low concentration. Here, we present a design of experiments (DOE) approach to generate samples for calibrating robust multivariate predictive models measuring glucose, lactate, ammonium, viable cell concentration (VCC) and product concentration, for unclarified cell culture that improves the daily monitoring of each miniature bioreactor vessel. Furthermore, we demonstrate how the output of the glucose and VCC models can be used to control the glucose and main nutrient feed rate within miniature bioreactor cultures to within qualified critical limits set for larger scale vessels. The DOE approach used to generate the calibration sample set is shown to result in models more robust to process changes than by simply using samples taken from the “typical” process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2740, 2019.     

Proteins can convert to beta-sheet in single crystals

Raman microscopy was used to follow conformational changes in single protein crystals. Crystals of native insulin and of the 5S and 12S subunits of the enzyme transcarboxylase showed a mixture of Raman marker bands signifying alpha-helix, beta-sheet and nonordered secondary structure. However, by reducing the S-S bonds in the insulin crystal, or by lowering the pH for the 5S crystal, or by soaking substrates into the 12S crystal, the secondary structure in each crystal became predominantly beta-sheet. The beta-form crystals could be dissolved only with difficulty and yielded high-molecular weight protein aggregates, indicating that the beta-sheet formation involves intermolecular contacts. Although their morphology appeared unchanged, the crystals no longer diffracted X-rays. Using crystals that had not been exposed to laser light, the dye thioflavin T formed highly fluorescent complexes with the "beta-transformed" crystals but not with the native crystals.     

Raman studies on bradykinin and a cyclic bradykinin

Laser Raman spectra of bradykinin in water, deuterium oxide, and the solid phase were recorded. From the spectra it was concluded that bradykinin conformation is comprised of ordered and unordered structure. The ordered structure appears to be some form of reverse turn. Furthermore, it seems that there is an enhancement of the turn structure in the solid phase. A cyclic cystine containing analog of bradykinin was also examined with Raman spectroscopy. The cyclic bradykinin analog gives a Raman spectrum very similar to that of the linear bradykinin and therefore must share similar conformational forms with bradykinin. The restrictive Cys-Cys disulfide in the cyclic bradykinin must serve to maintain a conformation acceptable to bradykinin receptors since the cyclic peptide exhibits biological activity.     

Autofluorescence and white light imaging‐guided endoscopic Raman and diffuse reflectance spectroscopy for in vivo nasopharyngeal cancer detection

Nasopharyngeal cancer (NPC) is an endemic with high incidence in Southern China and Southeast Asia countries. Screening for NPC under conventional white light imaging (WLI) nasopharyngoscope examination remains a great clinical challenge due to its poor sensitivity. Here, we developed an integrated 4‐modality endoscopy system combining WLI, autofluorescence imaging (AFI), diffuse reflectance spectroscopy and Raman spectroscopy technologies for in vivo endoscopic cancer detection for the first time. A pilot clinical test of the system for NPC detection was conducted, in which 283 in vivo Raman and diffuse reflectance spectral data sets from 30 NPC patients and 30 healthy subjects were acquired under the guidance of AFI and WLI. Both high diagnostic sensitivity (98.6%) and high specificity (95.1%) for differentiating cancer from normal tissue sites were achieved using this system combined with principal component analysis‐linear discriminant analysis diagnostic algorithm, demonstrating great potential for improving real‐time, in vivo diagnosis of NPC at endoscopy.      

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub‐organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live‐cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid‐based phenotypes.     

菜粉蝶颗粒体病毒分子与银表面间相互作用的SERS谱研究

通过对菜粉蝶颗粒体病毒粒子（样品）遥表面增强拉曼散射（ＳＥＲＳ）光谱的分析,得出样品分子在ＳＥＲＳ中与银表面相互作用的一些特征。样品分子主要通过闳基和氨基与银表面键联。相应谱带的拉曼增强效果与分子基因处于银表面的几何状态有关。分子与银表面产生化学吸附芳香族氨基酸侧链的π－电子得合物和分子基因的α－复合物的存在,使增强具有短程特性,其增强机制主要为分子增强。     

单细胞拉曼光谱在微生物研究中的应用

拉曼显微光谱是一种能够提供0.5–1.0μm空间分辨率的单个微生物细胞内化学结构信息的研究技术。近几年来,拉曼显微光谱被越来越多地应用于微生物单细胞的研究中,它可以快速无损地检测微生物细胞内的特征化学组分。典型的单个微生物细胞的拉曼光谱包含核酸、蛋白质、碳水化合物、脂质和色素(例如类胡萝卜素)等信息,这些信息能够表征微生物细胞的基因型、表型和生理状态。所以单细胞拉曼显微光谱是一种可用于区分微生物样品的"全生物指纹"技术,它可用于研究单个微生物细胞生命阶段的转变、鉴定微生物单细胞中的色素及其他化合物的含量变化等。本文综述了目前拉曼显微光谱在微生物单细胞研究上的应用,主要包括与稳定同位素标记(stable isotope probing,SIP)、拉曼成像、光谱分类和细胞分选技术结合来探究微生物单细胞对物质吸收后特征峰的变化、推导物质循环过程、进行微生物分类鉴定和探索基因型与表型的关系。拉曼显微光谱作为微生物单细胞研究的手段之一,在代谢过程的研究、活细胞分选和细胞对物质的利用上具有广泛的应用前景。     

Combining hyperspectral imaging and chemometrics to assess and interpret the effects of environmental stressors on zebrafish eye images at tissue level

Changes on an organism by the exposure to environmental stressors may be characterized by hyperspectral images (HSI), which preserve the morphology of biological samples, and suitable chemometric tools. The approach proposed allows assessing and interpreting the effect of contaminant exposure on heterogeneous biological samples monitored by HSI at specific tissue levels. In this work, the model example used consists of the study of the effect of the exposure of chlorpyrifos‐oxon on zebrafish tissues. To assess this effect, unmixing of the biological sample images followed by tissue‐specific classification models based on the unmixed spectral signatures is proposed. Unmixing and classification are performed by multivariate curve resolution‐alternating least squares (MCR‐ALS) and partial least squares‐discriminant analysis (PLS‐DA), respectively. Crucial aspects of the approach are: (1) the simultaneous MCR‐ALS analysis of all images from 1 population to take into account biological variability and provide reliable tissue spectral signatures, and (2) the use of resolved spectral signatures from control and exposed populations obtained from resampling of pixel subsets analyzed by MCR‐ALS multiset analysis as information for the tissue‐specific PLS‐DA classification models. Classification results diagnose the presence of a significant effect and identify the spectral regions at a tissue level responsible for the biological change.