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Analytical chemistry has never yielded such a wealth of experimental data as it does today, and this exponential trend shows no sign of abating. We continually advance the capabilities of our instruments and conceive innovative concepts, all in a concerted effort to naturally push the boundaries of our understanding regarding intricate sample matrices. Spectroscopic imaging, in the broadest sense, is certainly the field where we observe this acceleration even more pronouncedly. Analytical chemistry swiftly grasped the significance of processing acquired data for comprehensive exploration through utilization of chemometrics or machine learning tools. One can assert today that chemometrics undeniably constitutes an integral facet in the advancement of an analytical approach. However, we are now faced with a new challenge, as the experimental data accumulated for certain analytical techniques are so vast and massive that exploring them with such tools has become unfeasible, and this is by no means a computational capacity issue. Analytical chemistry is far from being the sole field affected by this issue, and one could argue that others have grappled with it long before us, such as, for instance, social media, to name just one. The purpose of this paper is to demonstrate that such a domain, which may initially seem distant from our concerns, can offer novel tools capable of overcoming these barriers, even though we are not necessarily dealing with the same objects. More specifically, we delve into the clustering of over 10 million LIBS spectra acquired as part of an imaging experiment aimed at exploring a singular rock sample. This will serve to demonstrate that an open-source library developed by Meta (formerly known as Facebook) can enable us to conduct a comprehensive exploration of this sample, a feat deemed impossible with conventional data analysis approaches.
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Laser-induced breakdown spectroscopy (LIBS) imaging continues to gain strength as an influential bioanalytical technique, showing intriguing potential in the field of clinical analysis. This is because hyperspectral LIBS imaging allows for rapid, comprehensive elemental analysis, covering elements from major to trace levels consistently year after year. In this study, we estimated the potential of a multivariate spectral data treatment approach based on a so-called convex envelope method to detect exotic elements (whether they are minor or in trace amounts) in biopsy tissues of patients with occupational exposure-related diseases. More precisely, we have developed an approach called Interesting Features Finder (IFF), which initially allowed us to identify unexpected elements without any preconceptions, considering only the set of spectra contained in a LIBS hyperspectral data cube. This task is, in fact, almost impossible with conventional chemometric tools, as it entails identifying a few exotic spectra among several hundred thousand others. Once this detection was performed, a second approach based on correlation was used to locate their distribution in the biopsies. Through this unique data analysis pipeline to processing massive LIBS spectroscopic data, it was possible to detect and locate exotic elements such as tin and rhodium in a patient's tissue section, ultimately leading to a possible reclassification of their lung condition as an occupational disease. This review will thus demonstrate the potential of this new diagnostic tool based on LIBS imaging in addressing the shortcomings of approaches developed thus far. The proposed data processing approach naturally transcends this specific framework and can be leveraged across various domains of analytical chemistry, where the detection of rare events is concealed within extensive data sets.
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Pneumopatias , Humanos , Biópsia , Pneumopatias/diagnóstico , Pneumopatias/patologia , Doenças Profissionais/diagnóstico , Doenças Profissionais/patologia , Lasers , Análise Espectral/métodos , Pulmão/patologia , Pulmão/química , Pulmão/diagnóstico por imagemRESUMO
In this study, we conducted a direct comparison of water-assisted laser desorption ionization (WALDI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging, with MALDI serving as the benchmark for label-free molecular tissue analysis in biomedical research. Specifically, we investigated the lipidomic profiles of several biological samples and calculated the similarity of detected peaks and Pearson's correlation of spectral profile intensities between the two techniques. We show that, overall, MALDI MS and WALDI MS present very close lipidomic analyses and that the highest similarity is obtained for the norharmane MALDI matrix. Indeed, for norharmane in negative ion mode, the lipidomic spectra revealed 100% similarity of detected peaks and over 0.90 intensity correlation between both technologies for five samples. The MALDI-MSI positive ion lipid spectra displayed more than 83% similarity of detected peaks compared to those of WALDI-MSI. However, we observed a lower percentage (77%) of detected peaks when comparing WALDI-MSI with MALDI-MSI due to the rich WALDI-MSI lipid spectra. Despite this difference, the global lipidomic spectra showed high consistency between the two technologies, indicating that they are governed by similar processes. Thanks to this similarity, we can increase datasets by including data from both modalities to either co-train classification models or obtain cross-interrogation.
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A total of 18 vacuum gas oils have been analyzed by Fourier transform ion cyclotron resonance mass spectrometry considering six replicates in three different ionization modes (electrospray ionization (ESI)(+), ESI(-), and atmospheric pressure photoionization (APPI)(+)) to characterize the nitrogen and sulfur compounds contained in these samples. Classical data analysis has been first performed on generated data sets using double bond equivalents (DBE) versus number of carbon atoms (#C) plots in order to observe similarities and differences within the nitrogen and sulfur-containing molecular classes from samples produced by different industrial processes. In a second step, three-way arrays have been generated for each ionization mode considering three dimensions: DBE related to aromaticity, number of carbon atoms related to alkylation, and sample. These three-way arrays have then be concatenated using low-level data fusion strategy to obtain a new tensor with three new modes: aromaticity, alkylation, and sample. The PARAFAC method has then been applied for the first time to this three-way data structure. A two components decomposition has allowed us to highlight unique samples with unexpected reactivity behaviors throughout hydrotreatment. The obtained loadings led to the identification of the variables responsible for this specific character. This original strategy has provided a fast visualization tool able to highlight simultaneously the impact of the three ionization modes in order to explain the differences between the samples and compare them.
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The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.
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Lignin is present in plant secondary cell walls and is among the most abundant biological polymers on Earth. In this work we investigated the potential role of the UGT72E gene family in regulating lignification in Arabidopsis. Chemical determination of floral stem lignin contents in ugt72e1, ugt72e2, and ugt72e3 mutants revealed no significant differences compared to WT plants. In contrast, the use of a novel safranin O ratiometric imaging technique indicated a significant increase in the cell wall lignin content of both interfascicular fibers and xylem from young regions of ugt72e3 mutant floral stems. These results were globally confirmed in interfascicular fibers by Raman microspectroscopy. Subsequent investigation using a bioorthogonal triple labelling strategy suggested that the augmentation in lignification was associated with an increased capacity of mutant cell walls to incorporate H-, G-, and S-monolignol reporters. Expression analysis showed that this increase was associated with an up-regulation of LAC17 and PRX71, which play a key role in lignin polymerization. Altogether, these results suggest that UGT72E3 can influence the kinetics of lignin deposition by regulating monolignol flow to the cell wall as well as the potential of this compartment to incorporate monomers into the growing lignin polymer.
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Proteínas de Arabidopsis/fisiologia , Arabidopsis , Parede Celular/metabolismo , Glucosiltransferases/fisiologia , Lignina/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Lignina/química , Mutação , Plantas Geneticamente Modificadas , Xilema/metabolismoRESUMO
Twenty-three gas oil samples from different origins were analyzed in positive and negative ion modes by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI(±)-FT-ICR MS). Sample ionization and ion transfer conditions were first optimized using Design of Experiment approach. Advanced characterization of basic and neutral nitrogen compounds in these samples was then performed through ESI(±)-FT-ICR MS analysis. A good repeatability was observed from the analysis of six replicates for each gas oil sample. Significant differences in molecular composition were spotted between the gas oils, either considering identified heteroatomic classes or within nitrogen families and were later correlated to samples macroscopic properties. The evolution of nitrogen relative intensities for one feed and two corresponding effluents has also been studied to monitor hydrotreatment reaction pathways toward aromaticity and alkylation levels evolutions.
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Sulfur content in gas oils is strictly regulated by legal specifications for environmental reasons. Gas oils are composed of various aromatic sulfur compounds, and some of them are known to be very refractory for sulfur removal reactions. Thus, an accurate analysis of sulfur compounds is important to find the appropriate operating conditions of the gas oil hydrotreating processes. Aromatic sulfur compounds contained in 23 gas oils samples were analyzed using APPI(+)-FT-ICR MS considering six replicates. Significant differences were spotted within several processed gas oils. A comparison of one feed and its corresponding effluents also confirmed the well-known refractory character of sulfur compounds such as polyalkylated dibenzothiophenes. To go deeper in the molecular exploration, chemometric tools were applied on this spectral data set including principal component analysis (PCA) and hierarchical cluster analysis (HCA). A unique data rearrangement was performed directly inspired on DBE vs carbon number plots that are systematically used in petroleomics studies. Then, these chemometric tools provided a successful classification of each type of gas oils. The PCA model has also been validated on mixed blends allowing us to conclude that it could be applied to unknown samples in order to identify the process used to produce them. Moreover, the exploration of the generated loadings revealed key types of molecules driving the classification such as C3-DBT which is a dibenzothiophene core with three additional carbon atoms. Indeed, it is known to remain mainly in deeply hydrotreated samples, validating previous observations regarding its potential refractory character. The ability of chemometric tools to extract specific molecular information from ultra-high resolution MS spectra reveals its huge potential for an exhaustive study of highly complex mixtures such as crude oils.
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Experimentally-generated (nanoLC-MS/MS) proteomic analyses of four different flax organs/tissues (inner-stem, outer-stem, leaves and roots) enriched in proteins from 3 different sub-compartments (soluble-, membrane-, and cell wall-proteins) was combined with publically available data on flax seed and whole-stem proteins to generate a flax protein database containing 2996 nonredundant total proteins. Subsequent multiple analyses (MapMan, CAZy, WallProtDB and expert curation) of this database were then used to identify a flax cell wall proteome consisting of 456 nonredundant proteins localized in the cell wall and/or associated with cell wall biosynthesis, remodeling and other cell wall related processes. Examination of the proteins present in different flax organs/tissues provided a detailed overview of cell wall metabolism and highlighted the importance of hemicellulose and pectin remodeling in stem tissues. Phylogenetic analyses of proteins in the cell wall proteome revealed an important paralogy in the class IIIA xyloglucan endo-transglycosylase/hydrolase (XTH) family associated with xyloglucan endo-hydrolase activity.Immunolocalisation, FT-IR microspectroscopy, and enzymatic fingerprinting indicated that flax fiber primary/S1 cell walls contained xyloglucans with typical substituted side chains as well as glucuronoxylans in much lower quantities. These results suggest a likely central role of xyloglucans and endotransglucosylase/hydrolase activity in flax fiber formation and cell wall remodeling processes.
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Parede Celular/metabolismo , Linho/metabolismo , Proteínas de Plantas/metabolismo , Polissacarídeos/metabolismo , Proteoma/metabolismo , Sequência de Aminoácidos , Epitopos/metabolismo , Funções Verossimilhança , Especificidade de Órgãos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Caules de Planta/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Following the Process Analytical Technology (PAT) of the Food and Drug Administration (FDA), drug manufacturers are encouraged to develop innovative techniques in order to monitor and understand their processes in a better way. Within this framework, it has been demonstrated that Raman spectroscopy coupled with chemometric tools allow to predict critical parameters of mammalian cell cultures in-line and in real time. However, the development of robust and predictive regression models clearly requires many batches in order to take into account inter-batch variability and enhance models accuracy. Nevertheless, this heavy procedure has to be repeated for every new line of cell culture involving many resources. This is why we propose in this paper to develop global regression models taking into account different cell lines. Such models are finally transferred to any culture of the cells involved. This article first demonstrates the feasibility of developing regression models, not only for mammalian cell lines (CHO and HeLa cell cultures), but also for insect cell lines (Sf9 cell cultures). Then global regression models are generated, based on CHO cells, HeLa cells, and Sf9 cells. Finally, these models are evaluated considering a fourth cell line(HEK cells). In addition to suitable predictions of glucose and lactate concentration of HEK cell cultures, we expose that by adding a single HEK-cell culture to the calibration set, the predictive ability of the regression models are substantially increased. In this way, we demonstrate that using global models, it is not necessary to consider many cultures of a new cell line in order to obtain accurate models. Biotechnol. Bioeng. 2017;114: 2550-2559. © 2017 Wiley Periodicals, Inc.
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Linhagem Celular/metabolismo , Glucose/metabolismo , Ácido Láctico/metabolismo , Análise do Fluxo Metabólico/métodos , Modelos Biológicos , Modelos Estatísticos , Animais , Simulação por Computador , Humanos , Insetos , Análise de RegressãoRESUMO
A calcium fluoride (CaF2) plate was exposed to pulsed laser irradiations inducing surface morphological and ionization changes on its surface. More precisely surface damages mainly correspond to intrinsic defects. Electron paramagnetic resonance (EPR) hyperspectral imaging is a powerful technique able to characterize the defects formed on the CaF2 surface. Indeed, EPR hyperspectral images provide spatial and spectral information about the sample studied. In fact, these images possess a great potential to obtain accurate and reliable knowledge about the chemical composition and the distribution of the component due to the presence of the spatial aspect. However, the complexity of such hyperspectral data sets imposes the use of advanced chemometric tools to extract valuable information on the considered physicochemical system. Therefore, Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) is proposed to identify and locate the different constituents in the images. The originality of this work is that it reports on the application of MCR-ALS, for the first time, on electron paramagnetic resonance (EPR) imaging data sets that will furnish the distribution maps and the spectral signatures of all components present in the sample. The results show the identification of different intrinsic defects on a CaF2 sample from the sole information in the raw image measurements and, therefore, confirm the potential of this methodology and the important role of spatial information contained in the image.
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Analytical chemistry on archaeological material is an essential part of modern archaeological investigations and from year to year, instrumental improvement has made it possible to generate data at a high spatial and temporal frequency. In particular, Raman spectral imaging can be successfully applied in archaeological research by its simplicity of implementation to study past human societies through the analysis of their material remains. This technique makes it possible to simultaneously obtain spatial and spectral information by preserving sample integrity. However, because of the inherent complexity of the samples in Archaeology (e.g. seniority, fragility, lack or full absence of any information about its composition), chemical interpretation can be difficult at first glance. Indeed, specific problems of spectral selectivity related to unexpected chemical compounds could appear due to their state of conservation. Furthermore, detecting minor compounds becomes challenging as major components impose their contributions in the acquired spectra. Therefore, a relevant chemometric approach has been introduced in this context to characterize distinct spectral sources in a Raman imaging dataset of an archaeological specimen - a mosaic fragment. The fragment was unearthed during the Ruscino archaeological dig on the outskirts of Perpignan, France. It dates back to the oppidum period. The aim is to extract selective spectral information from pixel clustering analysis in order to enhance the initial optimisation step within the Multivariate Curve Resolution and Alternating Least-Squares (MCR-ALS) algorithm, a well-known signal unmixing technique. The underlying principle of the MCR-ALS is that the acquired spectra can be expressed as linear combinations of pure spectra of all individual components present in the chemical system under study. Sometimes it can be difficult to obtain the desired results through the algorithm, particularly if initial estimates of spectral or concentration profiles are inaccurate due to complex signals, noise or lack of selectivity, resulting in rank deficiency (i.e. a poor estimation of the total number of pure signals). For this reason, an innovative threshold-based clustering algorithm, combined with multiple Orthogonal Projection Approaches (OPA), has been developed to improve matrix rank investigation and thus the initialisation step of the MCR-ALS approach before optimisation. The effective analysis of Raman imaging data for an archaeological mosaic played a crucial role in uncovering significant chemical information about a particular biogenic material. This insight sheds light on the origins of mortar manufacture during the oppidum period.
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Hyperspectral imaging technology is developing in a very fast way. We find it today in many analytical developments using different spectroscopies for sample classification purposes. Instrumental developments allow us to acquire more and more data in shorter and shorter periods of time while improving their quality. Therefore, we are going in the right direction as far as the measure is concerned. On the other hand, we can make a more mixed assessment for the hyperspectral imaging data processing. Indeed, the data acquired in spectroscopic imaging have the particularity of encoding both spectral and spatial information. Unfortunately, in chemometrics, almost all classification approaches today only use spectral information from three-dimensional hyperspectral data arrays. To be more precise, an approach encompassing the unfolding/refolding of such arrays is often applied beforehand because the majority of algorithms for analysing these data are not capable of handling them in their original structure. Spatial information is therefore lost during the chemometric exploration. The study of the spectral part of the acquired data array alone is clearly a limitation that we propose to overcome in this work. 2-D Stationary Wavelet Transform will be used in the data preprocessing phase to ensure the joint use of spectral and spatial information. Two spectroscopic datasets will then be used to evaluate the potential of our approach in the context of supervised classification.
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After death, diagenesis takes place. Numerous processes occur concomitantly, which makes it difficult to identify the diagenetic processes. The diagenetic processes refer to all processes (chemical or physical) that modify the skeletal remains. These processes are highly variable depending on the environmental factors (weather, temperature, age, sex, etc.), especially in the early stages. Numerous studies have evaluated bone diagenetic processes over long timescales (~millions of years), but fewer have been done over short timescales (between days and thousands of years). The objective of the study is to assess the early stages of diagenetic processes by Raman microspectroscopy over 12 months. The mineral and organic matrix modifications are monitored through physicochemical parameters. Ribs from six humans were buried in soil. The modifications of bone composition were followed by Raman spectroscopy each month. The decrease in the mineral/organic ratio and carbonate type-B content and the increase in crystallinity reveal that minerals undergo dissolution-recrystallization. The decrease in collagen cross-linking indicates that collagen hydrolysis induces the fragmentation of collagen fibres over 12 months.
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Laser-induced breakdown spectroscopy (LIBS) imaging is an innovative technique that associates the valuable atomic, ionic and molecular emission signals of the parent spectroscopy with spatial information. LIBS works using a powerful pulse laser as excitation source, to generate a plasma exhibiting emission lines of atoms, ions and molecules present in the ablated matter. The advantages of LIBS imaging are potential high sensitivity (in the order of ppm), easy sample preparation, fast acquisition rate (up to 1 kHz) and µm scale spatial resolution (weight of the ablated material in the order of ng). Despite these positive aspects, LIBS imaging easily provides datasets consisting of several million spectra, each containing several thousand spectral channels. Under these conditions, the current chemometric analyses of the raw data are still possible, but require too high computing resources. Therefore, the aim of this work is to propose a data compression strategy oriented to keep the most relevant spectral channel and pixel information to facilitate, fast and reliable signal unmixing for an exhaustive exploration of complex samples. This strategy will apply not only to the context of LIBS image analysis, but to the fusion of LIBS with other imaging technologies, a scenario where the data compression step becomes even more mandatory. The data fusion strategy will be applied to the analysis of a heterogeneous kyanite mineral sample containing several trace elements by LIBS imaging associated with plasma induced luminescence (PIL) imaging, these two signals being acquired simultaneously by the same microscope. The association of compression and spectral data fusion will allow extracting the compounds in the mineral sample associated with a fused LIBS/PIL fingerprint. This LIBS/PIL association will be essential to interpret the PIL spectral information, which is nowadays very complex due to the natural overlapped signals provided by this technique.
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Quimiometria , Luminescência , Lasers , Minerais , Análise EspectralRESUMO
The estimation of the postmortem interval (PMI) from skeletal remains represents a challenging task in forensic science. PMI is often influenced by extrinsic factors (humidity, dryness, scavengers, etc.) and intrinsic factors (age, sex, pathology, way of life, medical treatments, etc.). Raman spectroscopy combined with multivariate data analysis represents a promising tool for forensic anthropologists. Despite all the advantages of the technique, Raman spectra of skeletal remains are influenced by these extrinsic and intrinsic factors, which impairs precision and reproducibility. Both parameters have to reach a high level of confidence when such spectroscopy is used as a way to predict PMI. As a consequence, advanced multivariate data analysis is necessary to quantify the effect of all factors to improve the estimation of the PMI. The objective of this work is to evaluate the effect of intrinsic and extrinsic factors on the Raman spectra of skeletal remains. We designed a protocol close to a real-world scenario. We used ANOVA-simultaneous component analysis (ASCA) to unmix and quantify the effect of 1 intrinsic (source body) and 1 extrinsic (burial time) factors on the Raman spectra. In our model, the burial time was found to generate the highest variability after the source body. ASCA showed that the variability due to the burial time has 2 mixed contributions. Seasonal variations are the first contribution. The second contribution is attributed to diagenesis. A decrease in the mineral bands and an increase in the organic bands are observed. The source body was also found to contribute to the variability in Raman spectra. ASCA showed that the source body induces variability related to the composition of bones. This quantification cannot be assessed by basic chemometrics methods such as PCA. The results of this study highlighted the need to use an advanced chemometric data analysis tool (like ASCA) combined with Raman spectroscopy to estimate the postmortem interval.
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Restos Mortais , Análise Espectral Raman , Sepultamento , Humanos , Mudanças Depois da Morte , Reprodutibilidade dos Testes , Análise Espectral Raman/métodosRESUMO
Flax is an important fiber crop that is subject to lodging. In order to gain more information about the potential role of the bast fiber cell wall in the return to the vertical position, 6-week-old flax plants were subjected to a long-term (6 week) gravitropic stress by stem tilting in an experimental set-up that excluded autotropism. Stress induced significant morphometric changes (lumen surface, lumen diameter, and cell wall thickness and lumen surface/total fiber surface ratio) in pulling- and opposite-side fibers compared to control fibers. Changes in the relative amounts and spatial distribution of cell wall polymers in flax bast fibers were determined by Raman vibrational spectroscopy. Following spectra acquisition, datasets (control, pulling- and opposite sides) were analyzed by principal component analysis, PC score imaging, and Raman chemical cartography of significant chemical bonds. Our results show that gravitropic stress induces discrete but significant changes in the composition and/or spatial organization of cellulose, hemicelluloses and lignin within the cell walls of both pulling side and opposite side fibers.
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Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.
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Medula Óssea , Gotículas Lipídicas , Adulto , Humanos , Camundongos , Ratos , Animais , Coelhos , Camundongos Endogâmicos C57BL , Ácidos Graxos , Modelos Animais de DoençasRESUMO
Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.