Raman Microspectroscopy Research Articles

Microplastics evidence in yolk and liver of loggerhead sea turtles (Caretta caretta), a pilot study.

The potential toxicity of microplastics is a growing concern for the scientific community. The loggerhead sea turtle (Caretta caretta) is particularly inclined to accidently ingest plastic and microplastic due to its long-life cycle features. The possible transfer of microplastics from the female to the eggs should be investigated.The present study investigated the presence of microplastics in yolk and liver samples evaluating the number of melanomacrophages in the hepatic tissue as a possible biomarker of microplastics impact on the embryonic health status. The biometric parameters and liver histological analysis of 27 and 48 embryos (from two different nests respectively) at the 30 stage of development were analyzed. Raman Microspectroscopy was performed to identify the microplastics after alkaline digestion (10% KOH) of yolk and portion of liver from 5 embryos at the 30 developmental stage per nest. Microplastics were found in yolk and liver of loggerhead sea turtles at late embryonic stage for the first time. All microplastics were smaller than 5 μm and were made of polymers and colors suggesting their diverse origins. A total number of 21 microplastics, with dimensions lower than 5 μm, were found between the two nests (11 and 10 microplastics respectively). Only two shape categories were identified: spheres and fragments. The most frequent polymers observed were polyethylene, polyvinyl chloride and acrylonitrile butadiene styrene (31.5%, 21.1% and 15.8% respectively). Despite the eggs showing a higher number of microplastics in yolk samples than liver (15 and 6 microplastics in yolk and liver respectively), a positive correlation was observed only between the number of melanomacrophages (r = 0.863 p < 0.001) and microplastics in the liver. This result may suggest that microplastics could exert some effects on the hepatic tissues. Future studies should investigate this aspect and the possible relation between microplastics and other stress biomarkers.

Translating genomic tools to Raman spectroscopy analysis enables high-dimensional tissue characterization on molecular resolution

Spatial transcriptomics of histological sections have revolutionized research in life sciences and enabled unprecedented insights into genetic processes involved in tissue reorganization. However, in contrast to genomic analysis, the actual biomolecular composition of the sample has fallen behind, leaving a gap of potentially highly valuable information. Raman microspectroscopy provides untargeted spatiomolecular information at high resolution, capable of filling this gap. In this study we demonstrate spatially resolved Raman “spectromics” to reveal homogeneity, heterogeneity and dynamics of cell matrix on molecular levels by repurposing state-of-the-art bioinformatic analysis tools commonly used for transcriptomic analyses. By exploring sections of murine myocardial infarction and cardiac hypertrophy, we identify myocardial subclusters when spatially approaching the pathology, and define the surrounding metabolic and cellular (immune-) landscape. Our innovative, label-free, non-invasive “spectromics” approach could therefore open perspectives for a profound characterization of histological samples, while additionally allowing the combination with consecutive downstream analyses of the very same specimen.

Determination of atmospherically deposited microplastics in moss: Method development and performance evaluation

The investigation of atmospheric microplastic pollution is a rising topic within microplastic research. However, sampling strategies concerning atmospheric microplastics are not standardized yet and strongly depend on the scientific objectives investigated. A few research groups are currently focused on determining atmospherically deposited microplastics in moss, as it can be a biomonitoring system for other atmospheric pollutants such as heavy metals, nitrogen, and persistent organic components. In this context, and for atmospheric microplastics in general, almost all examinations focus on determining microplastic numbers and shapes, while polymer masses are barely investigated. However, particle- and mass-based information are needed to assess the fate of atmospherically transported microplastics in the environment. For this purpose, this study shows the development and evaluation of a new sample preparation method for determining microplastics in moss for both analytical approaches using thermal extraction desorption-gas chromatography-mass spectrometry (TED-GC-MS) and Raman microspectroscopy (µRaman). Further, this newly developed microplastics/moss separation method (µPEEL) was compared to oxidative digestion using Fenton's reagent, usually used as a sample preparation method for organic-rich samples and moss. The method comparison was performed concerning green analytical chemistry (GAC) and its respective method functionality. The greenness was assessed using the software tool AGREEprep, which covers ten comprehensive aspects of GAC. Both methods’ functionality was assessed regarding observable matrix interferences and the data quality achieved. It is demonstrated that µPEEL benefits GAC and method functionality since harmful chemicals can be avoided, matrix interferences can be minimized, and the separation quality is increased.

Optical Methods for Non-Invasive Determination of Skin Penetration: Current Trends, Advances, Possibilities, Prospects, and Translation into In Vivo Human Studies.

Information on the penetration depth, pathways, metabolization, storage of vehicles, active pharmaceutical ingredients (APIs), and functional cosmetic ingredients (FCIs) of topically applied formulations or contaminants (substances) in skin is of great importance for understanding their interaction with skin targets, treatment efficacy, and risk assessment-a challenging task in dermatology, cosmetology, and pharmacy. Non-invasive methods for the qualitative and quantitative visualization of substances in skin in vivo are favored and limited to optical imaging and spectroscopic methods such as fluorescence/reflectance confocal laser scanning microscopy (CLSM); two-photon tomography (2PT) combined with autofluorescence (2PT-AF), fluorescence lifetime imaging (2PT-FLIM), second-harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and reflectance confocal microscopy (2PT-RCM); three-photon tomography (3PT); confocal Raman micro-spectroscopy (CRM); surface-enhanced Raman scattering (SERS) micro-spectroscopy; stimulated Raman scattering (SRS) microscopy; and optical coherence tomography (OCT). This review summarizes the state of the art in the use of the CLSM, 2PT, 3PT, CRM, SERS, SRS, and OCT optical methods to study skin penetration in vivo non-invasively (302 references). The advantages, limitations, possibilities, and prospects of the reviewed optical methods are comprehensively discussed. The ex vivo studies discussed are potentially translatable into in vivo measurements. The requirements for the optical properties of substances to determine their penetration into skin by certain methods are highlighted.

Raman Diffusion-Ordered Spectroscopy.

The Stokes-Einstein relation, which relates the diffusion coefficient of a molecule to its hydrodynamic radius, is commonly used to determine molecular sizes in chemical analysis methods. Here, we combine the size sensitivity of such diffusion-based methods with the structure sensitivity of Raman spectroscopy by performing Raman diffusion-ordered spectroscopy (Raman-DOSY). The core of the Raman-DOSY setup is a flow cell with a Y-shaped channel containing two inlets: one for the sample solution and one for the pure solvent. The two liquids are injected at the same flow rate, giving rise to two parallel laminar flows in the channel. After the flow stops, the solute molecules diffuse from the solution-filled half of the channel into the solvent-filled half at a rate determined by their hydrodynamic radius. The arrival of the solute molecules in the solvent-filled half of the channel is recorded in a spectrally resolved manner by Raman microspectroscopy. From the time series of Raman spectra, a two-dimensional Raman-DOSY spectrum is obtained, which has the Raman frequency on one axis and the diffusion coefficient (or equivalently, hydrodynamic radius) on the other. In this way, Raman-DOSY spectrally resolves overlapping Raman peaks arising from molecules of different sizes. We demonstrate Raman-DOSY on samples containing up to three compounds and derive the diffusion coefficients of small molecules, proteins, and supramolecules (micelles), illustrating the versatility of Raman-DOSY. Raman-DOSY is label-free and does not require deuterated solvents and can thus be applied to samples and matrices that might be difficult to investigate with other diffusion-based spectroscopy methods.

It matters how we measure - Quantification of microplastics in drinking water by μFTIR and μRaman

The water treatment for microplastics (MP) at a Danish groundwater-based waterworks was assessed by Fourier-Transform IR micro-spectroscopy (μFTIR) (nominal size limit 6.6 μm) and compared to results from Raman micro-spectroscopy (μRaman) (nominal size limit 1.0 μm) on the same sample set. The MP abundance at the waterworks' inlet and outlet was quantified as MP counts per cubic metre (N/m3) and estimated MP mass per cubic metre (μg/m3). The waterworks' MP removal efficiency was found to be higher when analysing by μFTIR (counts: 78.14 ± 49.70%, mass: 98.73 ± 11.10%) and less fluctuating than when using μRaman (counts: 43.2%, mass: 75.1%). However, both techniques pointed to a value of ∼80% for the counts' removal efficiency of MPs >6.6 μm. Contrarily to what was shown by μRaman, no systematic leaking of MPs from the plastic elements of the facility could be identified for the μFTIR dataset, either from the counts (inlet 31.86 ± 17.17 N/m3, outlet 4.98 ± 2.09 N/m3) or mass estimate (inlet 76.30 ± 106.30 μg/m3, outlet 2.81 ± 2.78 μg/m3). The estimation of human MP intake from drinking water calculated from the μFTIR data (5 N/(year·capita)) proved to be approximately 332 times lower than that calculated from the μRaman dataset, although in line with previous studies employing μFTIR. By merging the MP length datasets from the two techniques, it could be shown that false negatives became prevalent in the μFTIR dataset already below 50 μm. Further, by fitting the overall frequency of the MP length ranges with a power function, it could be shown that μFTIR missed approximately 95.7% of the extrapolated MP population (1–1865.9 μm). Consequently, relying on only μFTIR may have led to underestimating the MP content of the investigated drinking water, as most of the 1–50 μm MP would have been missed.

Physiological and transcriptome profiling of Chlorella sorokiniana: A study on azo dye wastewater decolorization

Over decades, synthetic dyes have become increasingly dominated by azo dyes posing a significant environmental risk due to their toxicity. Microalgae-based systems may offer an alternative for treatment of azo dye effluents to conventional physical-chemical methods. Here, microalgae were tested to decolorize industrial azo dye wastewater (ADW). Chlorella sorokiniana showed the highest decolorization efficiency in a preliminary screening test. Subsequently, the optimization of the experimental design resulted in 70% decolorization in a photobioreactor. Tolerance of this strain was evidenced using multiple approaches (growth and chlorophyll content assays, scanning electron microscopy (SEM), and antioxidant level measurements). Raman microspectroscopy was employed for the quantification of ADW-specific compounds accumulated by the microalgal biomass. Finally, RNA-seq revealed the transcriptome profile of C. sorokiniana exposed to ADW for 72 h. Activated DNA repair and primary metabolism provided sufficient energy for microalgal growth to overcome the adverse toxic conditions. Furthermore, several transporter genes, oxidoreductases-, and glycosyltransferases-encoding genes were upregulated to effectively sequestrate and detoxify the ADW. This work demonstrates the potential utilization of C. sorokiniana as a tolerant strain for industrial wastewater treatment, emphasizing the regulation of its molecular mechanisms to cope with unfavorable growth conditions.

Dual-Stable-Isotope-Probed Raman microspectroscopy reveals the metabolic dynamic of Streptococcus mutans

Streptococcus mutans (S. mutans) is regarded as a cariogenic pathogen with the ability to metabolize sugars and form organic acids. However, its actual timely level of glucose consumption and cellular vitality in a polymicrobial culture system remains largely unknown. To tackle this challenge, we employed the S. mutans UA159 as a model and developed a dual-stable-isotope-probed Raman microspectroscopy method (Dual SIP-Raman) to simultaneously profile the general metabolic activity and glucose assimilative activity in situ. (i) Mono-SIP substrate feeding revealed that 0.5% 13C-glucose and 30% D2O were proper doses in the medium to obtain prominent and quantitative band shifts along with the 13C or D2O incorporation. In addition, the intensity of the 13C peak of phenylalanine (Phe) is proposed as a Raman-based biomarker for glucose utilization in a cell. (ii) The state of dual SIP substrate incorporation of 13C-glucose and D2O could be visualized by the corresponding spectral “red shifts” of Raman-scattered emissions; moreover, we also demonstrated that 13C/12C analysis was closely correlated with the C-D ratio. (iii) The application of the dual 13C-glucose and D2O feeding approach on a mock microbiota of S. mutans UA159 and C. albicans ATCC14053 revealed a stimulatory effect of fungus on both the glucose intake rate and general metabolic vitality of S. mutans UA159 (p < 0.05). Therefore, the 13C-glucose and D2O dual-feeding Raman Microspectroscopy approach is a valuable new tool for evaluating the glucose intake rate and general metabolic levels in situ, tracing the changing trend of the above metabolic activities, which is helpful to clarify the changes in the cariogenicity of oral microorganisms caused by the external environment at the single-cell level.

Topical dexpanthenol effects on physiological parameters of the stratum corneum by Confocal Raman Microspectroscopy.

Topical use of dexpanthenol presents well-established moisturizing properties and maintenance and repair of the skin barrier function, however, its exact action mechanisms are not completely elucidated. In this context, Confocal Raman Microspectroscopy is an optical method that enables non-invasive and non-destructive in vivo analysis with the sensitive acquisition of molecular changes in different skin layers. Herein, the aim was to evaluate the effects of topical dexpanthenol on the components and physiological parameters of the stratum corneum (SC). Ten healthy female subjects underwent skin evaluation by means of a Confocal Raman Spectrometer Skin Analyzer 3510. Spectral data were obtained from the skin of the anterior forearm region, before and 2h after applying a cosmetic formulation containing or not containing 5% dexpanthenol. Semiquantitative analysis of the natural moisturizing factor showed a significant decrease in content after 2 h of topical dexpanthenol application, while the analysis of the lamellar organization of intercellular lipids and the secondary structure of keratin showed a significant increase in hexagonal organization of lipids at the first half of the SC and a significant increase in β-pleated sheet conformation of keratin. Effects of topical dexpanthenol on SC suggest a contribution in increasing fluidity of both lipidic and protein components of the SC and are compatible with dexpanthenol activity in maintaining adequate physiological conditions and preventing transepidermal water loss. This study also contributes to the elucidation of action mechanisms and other concurrent biochemical processes.

Modeling versatile and dynamic anaerobic metabolism for PAOs/GAOs competition using agent-based model and verification via single cell Raman Micro-spectroscopy

Side-stream enhanced biological phosphorus removal process (S2EBPR) has been demonstrated to improve performance stability and offers a suite of advantages compared to conventional EBPR design. Design and optimization of S2EBPR require modification of the current EBPR models that were not able to fully reflect the metabolic functions of and competition between the polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) under extended anaerobic conditions as in the S2EBPR conditions. In this study, we proposed and validated an improved model (iEBPR) for simulating PAO and GAO competition that incorporated heterogeneity and versatility in PAO sequential polymer usage, staged maintenance-decay, and glycolysis-TCA pathway shifts. The iEBPR model was first calibrated against bulk batch testing experiment data and proved to perform better than the previous EBPR model for predicting the soluble orthoP, ammonia, biomass glycogen, and PHA temporal profiles in a starvation batch testing under prolonged anaerobic conditions. We further validated the model with another independent set of anaerobic testing data that included high-resolution single-cell and specific population level intracellular polymer measurements acquired with single-cell Raman micro-spectroscopy technique. The model accurately predicted the temporal changes in the intracellular polymers at cellular and population levels within PAOs and GAOs, and further confirmed the proposed mechanism of sequential polymer utilization, and polymer availability-dependent and staged maintenance-decay in PAOs. These results indicate that under extended anaerobic phases as in S2EBPR, the PAOs may gain competitive advantages over GAOs due to the possession of multiple intracellular polymers and the adaptive switching of the anaerobic metabolic pathways that consequently lead to the later and slower decay in PAOs than GAOs. The iEBPR model can be applied to facilitate and optimize the design and operations of S2EBPR for more reliable nutrient removal and recovery from wastewater.

(Invited) In-Situ Optical Diagnostics of Plasma Electrochemical Reactors

Many methods for synthesizing nanomaterials are complex and can require high temperature, acids, bases, or reducing agents. The plasma electrochemical reactor (PEC), composed of an atmospheric-pressure low-temperature plasma in contact with an aqueous electrode, may provide unique physico-chemical conditions that sidestep these disadvantages because non-equilibrium electrochemistry and nucleation are initiated in solution without additional heating or strong/toxic reagents. Indeed, PECs have successfully synthesized graphene quantum dots (GQD) [1,2].The precise mechanism of GQD growth is likely to be complex, involving non-equilibrium plasma chemistry and interactions near the plasma-liquid interface. Up to now, investigations of the liquid-phase chemistry have relied principally on ex situ measurements with little or no spatially-resolved information. In addition, conventional experimental techniques can suffer from a lack of selectivity and/or degradation of dyes, chemical probes, or spin traps introduced into the liquid.To extend the scope of diagnostics, we employ an in situ approach using multiple diagnostic techniques to study a wide range of physical and chemical properties at the plasma-water interface. The centrepiece of this platform is in situ spontaneous Raman microspectroscopy, which is advantageous because of its non-intrusiveness, selectivity, versatility, and straightforward calibration. Shaping the laser beam into a light sheet enables probing of the interfacial region with micron-scale spatial resolution. To observe the effect of the plasma on the solvent, we tracked the Raman spectrum of water. Analysis of the –OH stretch band reveals that the plasma weakens the hydrogen bonding network of water. This effect becomes especially pronounced near the interface, at a depth of a few tens of microns. Likewise, at a similar depth, the concentrations of aqueous H2O2 and NO3 - both exceed those of the bulk liquid [3]. Similar interfacial layers have been modeled in past studies for radical species such as OH but not for long-lived species such as NO3 -.Concerning GQDs, we tracked their production via in situ photoluminescence (PL) and UV-VIS absorption spectroscopies. Both the PL and absorption intensities reached a maximum not at the interface but rather at a depth of a few millimeters. This observation corroborates with liquid flow field measurements by particle image velocimetry, which revealed a low-velocity zone at this depth. Under certain conditions, we could observe the consumption of the carbon precursor over the course of plasma treatment using in situ Raman spectroscopy. Finally, we characterized plasma properties near the interface, such as the electron number density, using optical emission spectroscopy. Together, these results provide the fullest description to date of the reaction environment during GQD synthesis. Acknowledgments Financial support: ANR grants ANR-15-CE06-0007-01 and ANR-11-LABX-0017-01, PHC Orchid 40938YL, CNRS-IEA “GRAFMET”, Fédération Plas@Par, EUR PLASMAScience, Poitou-Charentes region (CPER program) References [1] Orrière, T., Kurniawan, D., Chang, Y. C., Pai, D. Z., & Chiang, W. H. (2020). Nanotechnology 31 (485001).[2] Yang, J. S., Pai, D. Z., & Chiang, W. H. (2019). Carbon 153, 315-319.[3] Pai, D. Z. (2021) J. Phys. D. : Appl. Phys. 54, 355201

Extracorporeal membrane oxygenation and effects on tendon tissue: A vibrational spectroscopy study.

Extracorporeal membrane oxygenation (ECMO) is an invasive medical technique used to provide life support in persons with insufficient cardiac and respiratory functionalities, or to preserve, postmortem, and organ function addressing organ/tissue transplant. Although a lot of information is available about organs in their entirety, the safety and effectiveness of allogeneic tissues collected from ECMO donors have not been fully elucidated. In this preliminary study, samples of tibial and peroneal human tendons were analyzed along their length with Raman microspectroscopy and attenuated total reflection-Fourier transform infrared micro-imaging. Both techniques evidenced a different chemical composition in the terminal with respect to the central part of the tendon. Thus, a differentiated analysis was performed depending on the specific position with respect to the bone or the muscle junctions. Spectroscopic analyses showed significant differences in the characteristics of the extracellular matrix between tendons from ECMO and non-ECMO donors, suggesting changes in the amino acid (proline and hydroxyproline) content and protein structure.

Crystallinity assessment of anthropogenic calcites using Raman micro-spectroscopy

Anthropogenic calcite is a form of calcium carbonate produced through pyrotechnological activities, and it is the main component of materials such as lime binders and wood ash. This type of calcite is characterized by a significantly lower degree of crystallinity compared with its geogenic counterparts, as a result of different formation processes. The crystallinity of calcite can be determined using infrared spectroscopy in transmission mode, which allows decoupling particle size effect from atomic order and thus effectively distinguish anthropogenic and geogenic calcites. On the contrary, Raman micro-spectroscopy is still in the process of developing a reference framework for the assessment of crystallinity in calcite. Band broadening has been identified as one of the proxies for crystallinity in the Raman spectra of geogenic and anthropogenic calcites. Here we analyze the full width at half maximum of calcite bands in various geogenic and anthropogenic materials, backed against an independent crystallinity reference based on infrared spectroscopy. Results are then used to assess the crystallinity of anthropogenic calcite in archaeological lime binders characterized by different states of preservation, including samples affected by the formation of secondary calcite, and tested on micromorphology thin sections in which lime binders are embedded in sediments.

Rapid identification of micro and nanoplastics by line scan Raman micro-spectroscopy

Microplastic pollution has become an environmental problem that cannot be ignored in our society. Raman spectroscopy technology has been widely used in the field of microplastics detection due to its non-contact, non-destructive chemical specificity. Traditional point confocal Raman micro-spectroscopy technology uses single-point detection, resulting in long measurement times to scan the large areas of interest of typical samples. In this paper, we present a line scan confocal Raman micro-spectroscopy tool for fast detection and identification of microplastic particles. We show size and composition identification of particles and imaging over large areas. Compared with point confocal Raman imaging, the line scan confocal Raman technology increases the imaging speed by 1–2 orders of magnitude.

Development of innovative antioxidant food packaging systems based on natural extracts from food industry waste and Moringa oleifera leaves

Active packaging that prolongs food shelf life, maintaining its quality and safety, is an increasing industrial demand, especially if integrated in a circular economy model. In this study, the fabrication and characterization of sustainable cellulose-based active packaging using food-industry waste and natural extracts as antioxidant agents was assessed. Grape marc, olive pomace and moringa leaf extracts obtained by supercritical fluid, antisolvent and maceration extraction in different solvents were compared for their antioxidant power and phenolic content. Grape and moringa macerates in acetone and methanol, as the most efficient and cost-effective extracts, were incorporated in the packaging as coatings or in-between layers. Both systems showed significant free-radical protection in vitro (antioxidant power 50%) and more than 50% prevention of ground beef lipid peroxidation over 16 days by indirect TBARS and direct in situ Raman microspectroscopy measurements. Therefore, these systems are promising for industrial applications and more sustainable farm-to-fork food production systems.

Altered structure indicating reduced barrier function of lesional compared to non-lesional psoriatic skin-A non-invasive in vivo study of the human stratum corneum with confocal Raman micro-spectroscopy.

Psoriasis, one of the most common skin diseases affecting roughly 2%-3% of the world population, is associated with a reduced skin barrier function (SBF) that might play an important role in its pathophysiology. The SBF is provided primarily by the stratum corneum (SC) of the skin. Previous studies have revealed a higher trans-epidermal water loss, lower hydration, abnormal concentration and composition of intercellular lipids, as well as alterations in secondary keratin structure in the psoriatic SC. We compared on molecular level lesional psoriatic skin (LPS) with non-lesional psoriatic skin (nLPS) from 19 patients non-invasively in vivo, using confocal Raman micro-spectroscopy. By analysing the corresponding Raman spectra, we determined SBF-defining parameters of the SC depth-dependently. Our results revealed a lower total lipid concentration, a shift of lamellar lipid organisation towards more gauche-conformers and an increase of the less dense hexagonal lateral packing of the intercellular lipids in LPS. Furthermore, we observed lower natural moisturising factor concentration, lower total water as well as a strong tendency towards less strongly bound and more weakly bound water molecules in LPS. Finally, we detected a less stable secondary keratin structure with increased β-sheets, in contrast to the tertiary structure, showing a higher degree of folded keratin in LPS. These findings clearly suggest structural differences indicating a reduced SBF in LPS, and are discussed in juxtaposition to preceding outcomes for psoriatic and healthy skin. Understanding the alterations of the psoriatic SC provides insights into the exact pathophysiology of psoriasis and paves the way for optimal future treatments.

Hyperglycemia exacerbates ischemic stroke not through increased generation of hydrogen peroxide

Diabetes is one of the significant risk factors for ischemic stroke. Hyperglycemia exacerbates the pathogenesis of stroke, leading to more extensive cerebral damage and, as a result, to more severe consequences. However, the mechanism whereby the hyperglycemic status in diabetes affects biochemical processes during the development of ischemic injury is still not fully understood. In the present work, we record for the first time the real-time dynamics of H2O2 in the matrix of neuronal mitochondria in vitro in culture and in vivo in the brain tissues of rats during development of ischemic stroke under conditions of hyperglycemia and normal glucose levels. To accomplish this, we used a highly sensitive HyPer7 biosensor and a fiber-optic interface technology. We demonstrated that a high glycemic status does not affect the generation of H2O2 in the tissues of the ischemic core, while significantly exacerbating the consequences of pathogenesis. For the first time using Raman microspectroscopy approach, we have shown how a sharp increase in the blood glucose level increases the relative amount of reduced cytochromes in the mitochondrial electron transport chain in neurons under normal conditions in awake mice.

Vibrational spectroscopy methods for investigation of the animal models of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and devastating primary brain tumor among adults. It is highly lethal disease, as only 25% of patients survive longer than 1 year and only 5% more than 5 years from the diagnosis. To search for the new, more effective methods of treatment, the understanding of mechanisms underlying the process of tumorigenesis is needed.The new light on this problem may be shed by the analysis of biochemical anomalies of tissues affected by tumor growth. Therefore, in the present work, we applied the Fourier transform infrared (FTIR) and Raman microspectroscopy to evaluate changes in the distribution and structure of biomolecules appearing in the rat brain as a result of glioblastoma development. In turn, synchrotron X-ray fluorescence microscopy was utilized to determine the elemental anomalies appearing in the nervous tissue. To achieve the assumed goals of the study animal models of GBM were used. The rats were subjected to the intracranial implantation of glioma cells with different degree of invasiveness. For spectroscopic investigation brain slices taken from the area of cancer cells administration were used.The obtained results revealed, among others, the decrease content of lipids and compounds containing carbonyl groups, compositional and structural changes of proteins as well as abnormalities in the distribution of low atomic number elements within the region of tumor.

Monitoring the macrophage response towards biomaterial implants using label-free imaging.

Understanding the immune system's foreign body response (FBR) is essential when developing and validating a biomaterial. Macrophage activation and proliferation are critical events in FBR that can determine the material's biocompatibility and fate in vivo. In this study, two different macro-encapsulation pouches intended for pancreatic islet transplantation were implanted into streptozotocin-induced diabetes rat models for 15 days. Post-explantation, the fibrotic capsules were analyzed by standard immunohistochemistry as well as non-invasive Raman microspectroscopy to determine the degree of FBR induced by both materials. The potential of Raman microspectroscopy to discern different processes of FBR was investigated and it was shown that Raman microspectroscopy is capable of targeting ECM components of the fibrotic capsule as well as pro and anti-inflammatory macrophage activation states, in a molecular-sensitive and marker-independent manner. In combination with multivariate analysis, spectral shifts reflecting conformational differences in Col I were identified and allowed to discriminate fibrotic and native interstitial connective tissue fibers. Moreover, spectral signatures retrieved from nuclei demonstrated changes in methylation states of nucleic acids in M1 and M2 phenotypes, relevant as indicator for fibrosis progression. This study could successfully implement Raman microspectroscopy as complementary tool to study in vivo immune-compatibility providing insightful information of FBR of biomaterials and medical devices, post-implantation.

Structure, microstructure and disorder in low temperature chemical vapor deposited SiC coatings

Chemical vapor deposited SiC coatings were investigated at different scales by X-Ray diffraction, Raman microspectroscopy and transmission electron microscopy. They were prepared under specific conditions explaining the various (micro)structures obtained. The deposits all have a columnar morphology with a preferential orientation and a faulted cubic structure. They differ in how disorder is incorporated in the structure. Fine XRD analyses and stacking fault density assessment by TEM revealed the one-dimensionally-disordered (ODD) polytype in the <111> textured coatings. The frequency and spatial distribution of stacking faults vary and sometimes locally generate periodic alpha sequences. A specific type of disorder was also identified where {111} planes are arranged parallel to the growth direction within the columns. These disorders, more energetic than stacking faults, induce multiple and particularly large Raman modes. Crystal distortions, such as dislocations, are localized at the ODD domain boundaries, which are frequently interrupted as they extend during the growth.