Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Preparation of soybean seed samples for FT-IR microspectroscopy.

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4 degrees C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Classification and identification of Arabidopsis cell wall mutants using Fourier-Transform InfraRed (FT-IR) microspectroscopy

We have developed a novel procedure for the rapid classification and identification of Arabidopsis mutants with altered cell wall architecture based on Fourier-Transform Infrared (FT-IR) microspectroscopy. FT-IR transmission spectra were sampled from native 4-day-old dark-grown hypocotyls of 46 mutants and the wild type treated with various drugs. The Mahalanobis distance between mutants, calculated from the spectral information after compression with the Discriminant Variables Selection procedure, was used for alpha hierarchical cluster analysis. Despite the completely unsupervised nature of the classification procedure, we show that all mutants with cellulose defects appeared in the same cluster. In addition, mutant alleles of similar strength for several unrelated loci were also clustered, which demonstrates the sensitivity of the method to detect a wide array of cell wall defects. Comparing the cellulose-deficient cluster with the cluster that contained wild-type controls led to the identification of wave numbers that were diagnostic for altered cellulose content in the context of an intact cell wall. The results show that FT-IR spectra can be used to identify different classes of mutants and to characterize cell wall changes at a microscopic level in unknown mutants. This procedure significantly accelerates the identification and classification of cell wall mutants, which makes cell wall polysaccharides more accessible to functional genomics approaches.     

In-vitro analysis of normal and aneurismal human ascending aortic tissues using FT-IR microspectroscopy

FTIR microspectroscopy has shown to be a proven tool in the investigation of many tissue types. We have used this spectroscopic approach to analyse structural differences between normal and aneurismal aortic tissues and also aortas from patients with congenital anomalies like aortic bicuspid valves. Spectral analysis showed important variations in amide I and II regions, related to changes in alpha-helix and beta-sheet secondary structure of proteins that seem to be correlated to structural modifications of collagen and elastin. These proteins are the major constituents of the aortic wall associated to smooth muscular cells. The amide regions have thus been identified as a marker of structural modifications related to these proteins whose modifications can be associated to a given aortic pathological situation. Both univariate (total absorbance image and band ratio) and multivariate (principal components analysis) analyses of the spectral information contained in the infrared images have been performed. Differences between tissues have been identified by these two approaches and allowed to separate each group of aortic tissues. However, with univariate band ratio analysis, the pathological group was found to be composed of samples from aneurismal aortas associated or not with an aortic bicuspid valve. In contrast, PCA was able to separate these two types of aortic pathologies. For other groups, PCA and band ratio analysis can differentiate between normal, aneurismal, and none dilated aortas from patients with a bicuspid aortic valve.     

FT-IR microspectroscopy: a promising method for the rapid identification of Listeria species

This work presents a pilot study to investigate the potential of fourier transform infrared (FT-IR) microspectroscopy for rapid identification of Listeria at the species level. Using this technique, FT-IR spectra were acquired from 30 strains from five Listeria species. The FT-IR spectra were analysed using stepwise canonical discriminant analysis and partial least-squares regression in a stepwise identification scheme. The results showed that 93% of all the samples were assigned to the correct species, and that 80% of the Listeria monocytogenes strains were correctly identified. In comparison, 100% of the samples, including the L. monocytogenes samples, were correctly identified using spectra acquired by FT-IR macrospectroscopy. The results show that FT-IR microspectroscopy has potential as a rapid screening method for Listeria, which is especially valuable for the food industry.     

Variability of protein and lipid composition of human subtantia nigra in aging: Fourier transform infrared microspectroscopy study

There is growing evidence that a variety of biochemical processes that underlie the most frequent neurodegenerative diseases may have much in common with those connected with natural aging. It was shown that they involve, among others, lipid peroxidation and/or generation of insoluble in water protein deposits (i.e. alpha-synuclein and/or beta amyloid). Therefore, it is likely that the analysis of changes in both lipid and protein composition may be interesting in the light of any potential pathologies occurring within the dopaminergic system during physiological aging. Thereby, this paper presents a methodology for the analysis of age-related changes in a lipid and protein composition within human subtantia nigra tissue by means of Fourier transform infrared microspectroscopy (FTIRM). Particularly, the changes in the lipid saturation, unsaturation as well as in the protein secondary structure were examined. The studies were carried out on samples from 35 individuals who died without any signs of neurologic dysfunctions. Our results show that the level of lipid saturation increases inside the subtantia nigra tissue with age, though the total content of lipid decreases with age of individuals. Moreover, the statistically significant decrease in the protein content within neuron bodies was observed. Interestingly, it is presented that the content of the anti-parallel beta sheets for neuron bodies decreases from seventh to eighth decades of life and subsequently markedly increases from eighth to ninth decades of life, whilst, as regards extraneuronal spaces, the opposite trends are reported i.e. increase from the seventh to eighth decades, and subsequent decrease in the ninth decade of life. These observations, though preliminary, shed the light on a putative contribution of various pathological lipid- and protein-related processes underlying senescence, suggesting a “biochemical link” between the aetiology of the most common neurodegenerative diseases and physiological aging.     

Fourier transform infrared microspectroscopy as a new tool for nematode studies

We report the results of a microspectroscopy study on the Fourier transform infrared (FT-IR) absorption spectra of Caenorhabditis elegans, collected from the different parts of a single intact specimen--pharynx, intestine and tail regions. The principal absorption bands were assigned to the molecular species present in C. elegans, with an excellent reproducibility for the pharynx spectrum. These results enabled us to explore if FT-IR microspectroscopy could offer a new tool for nematode identification. As an example, the discrimination among four well characterised nematode taxa is reported. The FT-IR results completely match those obtained by Blaxter and colleagues through molecular biology [Nature 392 (1998) 71].     

Study of tumor cell invasion by Fourier transform infrared microspectroscopy

Lung cancer is usually fatal once it becomes metastatic. However, in order to develop metastases, a tumor usually invades the basal membrane and enters the vascular or lymphatic system. In this study, a three-dimensional artificial membrane using collagen type I, one of the main components of basal membranes, was established in order to investigate tumor cell invasion. Lung cancer cell line CALU-1 was seeded on this artificial membrane and cell invasion was studied using the Fourier transform infrared (FTIR) imaging technique. This approach allowed identification of tumor cells invading the collagen type I membrane by means of their infrared spectra and images. The mapping images obtained with FTIR microspectroscopy were validated with standard histological section analysis. The FTIR image produced using a single wavenumber at 1080 cm(-1), corresponding to PO2- groups in DNA from cells, correlated well with the histological section, which clearly revealed a cell layer and invading cells within the membrane. Furthermore, the peaks corresponding to amide A, I, and II in the spectra of the invading cells shifted compared to the noninvading cells, which may relate to the changes in conformation and/or heterogeneity in the phenotype of the cells. The data presented in this study demonstrate that FTIR microspectroscopy can be a fast and reliable technique to assess tumor invasion in vitro.     

On the role of interference in laser‐based mid‐infrared widefield microspectroscopy

A laser's high degree of coherence leads to interferences, which—in the absence of precautions—can cause severe image distortions such as fringes and speckles and which thereby strongly hamper a meaningful interpretation of hyperspectral images in laser‐based widefield microspectroscopy. While images and spectra of homogenous samples may already suffer from interferences, any structured object such as a tissue thin section will add to these distortions due to wavelength‐ and, in particular, sample‐dependent phase shifts (structure sizes, absorption coefficients, refractive indices). This effect is devastating for the universal applicability of laser‐based microspectroscopy especially in the mid‐infrared (MIR), where cell sizes are of the same dimension as the wavelength of the illumination source. Here, we show that the impact of interferences is strongly mitigated by reducing the time‐averaged spatiotemporal coherence properties of the illumination using a moving plus a stationary scatterer. In this case, the illumination path provides a pseudothermal radiation source and spatially resolved spectra can be obtained at the quality of the reference method, that is, Fourier‐transform infrared microspectroscopy, without compromising spectral or spatial resolution.      

Polarized infrared microspectroscopy of single spruce fibers: hydrogen bonding in wood polymers

We studied wood polymers in their native composite structure using mechanically isolated single spruce (Picea abies [L.] Karst.) fibers. Dichroic infrared spectra of fibers placed in a custom-built microfluidic cuvette were acquired in air, in liquid (heavy) water, and in liquid dimethylacetamide using a novel combination of synchrotron-based Fourier transform infrared microspectroscopy with polarization modulation. Differences were observed in the O-H stretching frequency region of the spruce spectra upon changing the ambient conditions. Analysis of these spectral variations provides information on hydrogen bonding, orientation, and accessibility of structural units of the wood polymers in the spruce cell walls. Our in situ approach contributes to a further understanding of the structural details of wood polymers in their native setting.     

Fourier transform infrared microspectroscopy reveals unique phenotypes for human embryonic and induced pluripotent stem cell lines and their progeny

Fourier transform infrared (FTIR) microspectroscopy was employed to elucidate the macromolecular phenotype of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and their differentiated progeny. Undifferentiated hESCs and hiPSC lines were found to be not clearly distinguishable from each other. However, although both hESC and hiPSC variants appeared to undergo similar changes during differentiation in terms of cell surface antigens, the derived cell types from all cell lines could be discriminated using FTIR spectroscopy. We foresee a possible future role for FTIR microspectroscopy as a powerful and objective investigative and quality control tool in regenerative medicine. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

FT-IR microspectroscopy for early identification of some clinically relevant pathogens

AIMS: To investigate the potentials and limitations of Fourier transform-infrared (FT-IR) microspectroscopy as a tool to identify, at the level of microcolonies, pathogenic bacteria frequently isolated in the clinical environment. METHODS AND RESULTS: A total of 1570 FT-IR spectra from 164 gram-positive and gram-negative bacteria isolated from patients were recorded from 6 to 10-h old microcolonies of 50-150 microm size. A classification of 100% was obtained for the most frequent gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium at the species level. An average accuracy of about 80% was reached with Gram negative bacteria from the Enterobacteriaceae and Pseudomonaceae families; Enterobacter aerogenes, Enterobacter cloacae, Klebsiella spp., and Citrobacter koseri; and Proteus mirabilis and Escherichia coli. Results were comparable with FT-IR measurements on dried suspensions from 18-h cultures. CONCLUSIONS: Early identification of young microcolonies is feasible with FT-IR microscopy with a very high accuracy for gram-positive bacteria. Some improvement in the transfer of microcolonies is necessary to increase the accuracy for gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Combination of FT-IR microscopy and multivariate data analysis could be a complementary, rapid, and reliable tool for screening and discriminating, at species and subspecies level, micro-organisms of clinical, food-borne, or environmental origins.     

Cell-specific chemotyping and multivariate imaging by combined FT-IR microspectroscopy and orthogonal projections to latent structures (OPLS) analysis reveals the chemical landscape of secondary xylem

Fourier‐transform infrared (FT‐IR) spectroscopy combined with microscopy enables chemical information to be acquired from native plant cell walls with high spatial resolution. Combined with a 64 × 64 focal plane array (FPA) detector, 4096 spectra can be simultaneously obtained from a 0.3 × 0.3 mm image; each spectrum represents a compositional and structural ‘fingerprint’ of all cell wall components. For optimal use and analysis of such a large amount of information, multivariate approaches are preferred. Here, FT‐IR microspectroscopy with FPA detection is combined with orthogonal projections to latent structures discriminant analysis (OPLS‐DA). This allows for: (i) the extraction of spectra from single cell types, (ii) identification and characterization of different chemotypes using the full spectral information, and (iii) further visualization of the pattern of identified chemotypes by multivariate imaging. As proof of concept, the chemotypes of Populus tremula xylem cell types are described. The approach revealed unknown features about chemical plasticity and patterns of lignin composition in wood fibers that would have remained hidden in the dataset with traditional data analysis. The applicability of the method to Arabidopsis xylem and its usefulness in mutant chemotyping is also demonstrated. The methodological approach is not limited to xylem tissues but can be applied to any plant organ/tissue also using other techniques such as Raman and UV microspectroscopy.     

Concepts and strategies of soybean seed proteomics using the shotgun proteomics approach

ABSTRACT

Introduction: The last decade has yielded significant developments in the field of proteomics, especially in mass spectrometry (MS) and data analysis tools. In particular, a shift from gel-based to MS-based proteomics has been observed, thereby providing a platform with which to construct proteome atlases for all life forms. Nevertheless, the analysis of plant proteomes, especially those of samples that contain high-abundance proteins (HAPs), such as soybean seeds, remains challenging.

Areas covered: Here, we review recent progress in soybean seed proteomics and highlight advances in HAPs depletion methods and peptide pre-fractionation, identification, and quantification methods. We also suggest a pipeline for future proteomic analysis, in order to increase the dynamic coverage of the soybean seed proteome.

Expert opinion: Because HAPs limit the dynamic resolution of the soybean seed proteome, the depletion of HAPs is a prerequisite of high-throughput proteome analysis, and owing to the use of two-dimensional gel electrophoresis-based proteomic approaches, few soybean seed proteins have been identified or characterized. Recent advances in proteomic technologies, which have significantly increased the proteome coverage of other plants, could be used to overcome the current complexity and limitation of soybean seed proteomics.     

Comparison of methods for sample preparation of individual rat cerebrospinal fluid samples prior to two-dimensional polyacrylamide gel electrophoresis

Different pre-treatment methods have been compared for two-dimensional mapping of individual rat cerebrospinal fluid samples based on acetone, trichloroacetic acid/acetone and methanol/acetone precipitation of proteins. Acetone precipitation following incubation with DTT gave the highest protein recovery (72%) and the largest number of protein spots (92 +/- 4) as well as minimizing the time taken.     

有鳞目动物显微皮纹学研究样品制备方法

有鳞目动物具有复杂的蜕皮循环,表皮层还具有复杂的组织学层次,然而只有最外面的角皮层有稳定的微细结构.在扫描电镜样品的制备过程中,常因不能区别蜕皮循环阶段造成混淆组织层次而影响研究成果的准确性.本文介绍的整体切割样品制备技术可以估计蜕皮循环阶段而无需依赖组织学技术,还介绍了一种简单的光学显微镜下皮纹学装片制作方法.     

Biomolecular investigation of human substantia nigra in Parkinson's disease by synchrotron radiation Fourier transform infrared microspectroscopy

Synchrotron radiation based-Fourier transform infrared microspectroscopy was used for preliminary investigation of the chemical composition and morphologies of the human substantia nigra of brain between normal and Parkinson's diseased tissues. The studies were carried out for thin tissue sections, focusing more particularly on nerve cell bodies, that are affected in Parkinson's disease (PD). The major spectral differences between normal (control) and PD tissues were identified at the following vibrational frequencies: 2930, 2850, 1655, 1380, 1236, 1173 and 1086 cm(-1). The infrared imaging of these biochemical markers show that for control cases the protein and nucleic acids functional groups (bands at: approximately 3300, approximately 3100, approximately 1655, approximately 1545, approximately 1240, approximately 1080 cm(-1)) are located mainly in the cell body. The spatial distribution of the band at 1740 cm(-1) (ester carbonyl stretching band) is quite dissimilar to the others, while it exhibits a minimal concentration in the cell body area. Contrarily, in PD samples, no clear evidence of variation of any of the vibrational fingerprint between cell body and the surrounding was noticed. Moreover, decrease of protein to lipid ratio as well as increase of amide I/amide II ratio were observed for PD case. The preliminary results strengthen the hypothesis that PD is a multietiological disorder. Moreover, the reported results clearly indicate that, in addition to a distinct visual observation, the diseased nerve cells exhibits change of their biochemical composition. It suggests that disturbances of normal functioning of SN neurons appear before their morphological atrophy.     

用于金刚石表面单分子有序固定的DNA折纸制备

近年来兴起的以金刚石氮-空位(NV)色心为量子传感器的微观磁共振技术得到快速发展,已经实现单个生物分子磁共振谱的探测,正在向单分子结构和功能的研究推进.这其中需要解决一个重要的技术问题,即单分子在金刚石表面的有序分散和固定. DNA的自组装为解决这一问题提供了可行途径,本文使用DNA折纸技术,制备了一种60 nm边长的正方形双层DNA折纸作为单分子载体,并与金刚石表面结合.首先采用双层结构提高了DNA折纸的稳定性,其次通过在DNA折纸边缘添加发卡结构减少了DNA折纸结构间的聚团,最终成功将DNA折纸装配到金刚石表面.通过原子力显微镜图像进行表征显示其结构完整、分散均匀.本工作为后续的单分子磁共振技术在单分子生物物理领域的应用推广奠定了样品制备的基础.     

基于超滤辅助样品制备方法的优化简

目的：基于超滤辅助样品制备（FASP）方法的出现使得使用去污剂（如SDS）的蛋白质提取方法与溶液内酶切方法得以兼容,因此提高了难溶性蛋白的鉴定数量。然而,超滤膜的非特异性吸附作用依然会造成蛋白的损失。我们拟针对该方法存在的问题对其进行改进。方法：对FASP方法进行了蛋白酶切条件、洗脱液选择、洗脱次数的改进;为测试优化方法的有效性和适用范围,选择标准蛋白BSA、鼠肝和鼠脑等3种样品进行考察。结果：相比报道的FASP方法,采用改进后的FASP方法使BSA的回收率提高了20％;经高精度质谱检测,对鼠肝、鼠脑的蛋白质鉴定结果分别比采用未优化的FASP多鉴定到2086和3592条特异肽段。结论：通过对FASP方法的优化,蛋白鉴定数量得到较大提高,该方法为蛋白质组深度覆盖研究提供了可靠的技术手段。     

Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ

AIMS: To investigate the use of confocal Raman microspectroscopy (CRM) for the analysis of the structure, composition and development of fully hydrated biofilms. METHODS AND RESULTS: Pseudomonas aeruginosa PAO1 biofilms were cultured in a flow cell in minimal nutrient medium (artificial sea water) and their development was followed for up to 3 weeks. The spectroscopic signature of the biofilm cells and extracellular polymeric substances (EPS) were differentiated and their distribution in biofilm colonies and within water channels was mapped in-plane and -depth. The colonies were initially amorphous, mainly composed of cells with no detectable amount of EPS. They developed rapidly to give round colonies composed of a cellular core enclosed in a sheath of EPS. The EPS continued to increase and spread throughout the biofilm to become the dominating feature of aged colonies. Colonies with a liquid core morphology - characteristic of the seeding dispersal process - were also observed. CONCLUSIONS: This study demonstrated that CRM can be used to monitor the distribution of biofilm components in fully hydrated undisturbed biofilms over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Confocal Raman microspectroscopy facilitates the analysis of hydrated, live bacterial biofilms as a function of space and time, thus making it a suitable technique for investigating the effects of various additives and environmental factors on biofilm growth.