Raman Microspectroscopy Analysis Research Articles

A Study on the Corrosion and Microstructure Characteristics of Early Iron Age Bronze Swords Excavated from Sinpung Site in Wanju, Jeollabuk-Do, Republic of Korea

The subject of this study was a bronze sword excavated from the early Iron Age area of Sinpung site in Wanju. SEM-EDS, FE-EPMA and Raman Micro-Spectroscopy analysis methods were applied to investigate the manufacturing techniques and corrosion characteristics. The results of the analysis indicated a casting structure composed of both α and α+δ phases. The α phase developed in the form of dendrite, and it seems that no heat treatment was performed. Through the EDS analysis results, the main components of the bronze sword were identified as Cu, Sn, Pb, etc., and it seems that the cooling rate was different depending on the shape of the dendrite, which is the α phase, and the size and distribution of the Pb particles. Regarding corrosion characteristics, the corrosion progressed considerably to the surface, and a high tin corrosion layer was formed, and lead oxide and copper oxide particles were observed between the corroded microstructures. These materials were identified by Raman spectroscopic analysis as Cuprite (Cu2O), Malachite (Cu2CO3(OH)2), Cassiterite (SnO2), Cerussite (PbCO3), Anglesite (PbSO4), and Litharge (PbO). Among them, the presence of nanocrystalline SnO2 confirmed the characteristics of long-term corrosion of bronze artifacts in the burial environment. And, materials such as Cerussite (PbCO3), Anglesite (PbSO4), and Litharge (PbO) were found only in weapons and tools such as bronze sword and spear when compared with previous studies.

Atomic Force Microscopy and Raman Microspectroscopy Investigations of the Leaching of Chalcopyrite (112) Surface

The aim of this study was to determine the reactivity of the chalcopyrite (112) surface under industrially relevant leaching conditions. Leaching of the chalcopyrite (112) surface was carried out at approximately pH 1 and in the presence of 0.01 M ferric or ferrous. The atomic force microscopy (AFM) and Raman microspectroscopy analyses suggested that the chalcopyrite (112) surface was relatively inert, with no formation of elemental sulfur observed over 42 days of leaching. In addition, it was found that the distribution of Fe-S and Cu-S bonds was always negatively correlated, as revealed by Raman analysis. This suggested that the breakage of the Fe-S and Cu-S bonds did not occur concurrently at a specific reaction site. The rate of variation of surface roughness, as reflected by AFM data, also suggested that leaching of the chalcopyrite (112) surface in the ferric or ferrous solution medium likely occurred more rapidly in the initial stage (fewer than seven days) than in the later stage (after seven days).

Confocal Raman microspectroscopic analysis on the time-dependent impact of DAPT, a γ-secretase inhibitor, to osteosarcoma cells.

Confocal Raman microspectroscopy (CRM) analysis provides subcellular compositional and morphology related information. In this study, we used CRM in conjunction with multivariate statistical analysis to elucidate the time-dependent impact of the γ-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) of osteosarcoma (OS) cells. The interactions of DAPT (20μM) with a murine OS cell line K7M2 at 24 and 48h were monitored. The spectral characteristics of drug action were identified to illustrate the cellular compositional alterations, showing that DAPT induced apoptosis by reducing the protein, lipid and nucleic acid content and structural changes. Multivariate algorithms, principal component analysis (PCA) and linear discriminant analysis (LDA) revealed a clear separation among cells in the untreated control (UT), 24H (DAPT-treated for 24h), and 48H (DAPT-treated for 48h) groups, achieving sensitivities of 100%, 96%, 100% and specificities of 98%, 100%, 100%, respectively. After point-scanned spectral imaging, K-means clustering analysis (KCA) was further used to visualize sub-cellular morphological changes and the underlying spectral characteristics in a temporal sequence. Compared with the UT group, Raman imaging results exhibited gradually increased nuclear division of OS cells with DAPT treatment duration extension, along with changes in the physiology of other organelles within the cell. By providing a unique perspective for understanding the temporary cellular responses to DAPT at molecular level, the achieved results form the foundation of strategies for the application of CRM and other Raman-based techniques for studying the therapeutic responses of other anticancer agents in cancer model systems.

Translation Approach for Dentine Regeneration Using GSK-3 Antagonists

The canonical Wnt/β-catenin signaling pathway is crucial for reparative dentinogenesis following tooth damage, and the modulation of this pathway affects the rate and extent of reparative dentine formation in damaged mice molars by triggering the natural process of dentinogenesis. Pharmacological stimulation of Wnt/β-catenin signaling activity by small-molecule GSK-3 inhibitor drugs following pulp exposure in mouse molars results in reparative dentinogenesis. The creation of similar but larger lesions in rat molars shows that the adenosine triphosphate (ATP)–competitive GSK-3 inhibitor, CHIR99021 (CHIR), and the ATP noncompetitive inhibitor, Tideglusib (TG), can equally enhance reparative dentine formation to fully repair an area of dentine damage up to 10 times larger, mimicking the size of small lesions in humans. To assess the chemical composition of this newly formed dentine and to compare its structure with surrounding native dentine and alveolar bone, Raman microspectroscopy analysis is used. We show that the newly formed dentine comprises equal carbonate to phosphate ratios and mineral to matrix ratios to that of native dentine, both being significantly different from bone. For an effective dentine repair, the activity of the drugs needs to be restricted to the region of damage. To investigate the range of drug-induced Wnt-activity within the dental pulp, RNA of short-term induced (24-h) molars is extracted from separated roots and crowns, and quantitative Axin2 expression is assayed. We show that the activation of Wnt/β-catenin signaling is highly restricted to pulp cells in the immediate location of the damage in the coronal pulp tissue with no drug action detected in the root pulp. These results provide further evidence that this simple method of enhancement of natural reparative dentinogenesis has the potential to be translated into a clinical direct capping approach.

Raman microspectroscopic analysis of the tissue-specific composition of the human osteochondral junction in osteoarthritis: A pilot study

This study investigates the influence of osteoarthritis (OA) disease severity on the bio-composition of the osteochondral junction at the human tibial plateau using Raman microspectroscopy. We specifically aim to analyze the spatial composition of mineralized osteochondral tissues, i.e., calcified cartilage (CC) and subchondral bone plate (SBP) from unfixed, hydrated specimens. We hypothesize that the mineralization of CC and SBP decreases in advanced OA. Twenty-eight cylindrical osteochondral samples (d = 4 mm) from tibial plateaus of seven cadaveric donors were harvested and sorted into three groups following histopathological grading: healthy (n = 5), early OA (n = 8), and advanced OA (n = 15). Raman spectra were subjected to multivariate cluster analyses to identify different tissues. Finally, the tissue-specific composition was analyzed, and the impact of OA was statistically evaluated with linear mixed models. Cluster analyses of Raman spectra successfully distinguished CC and SBP as well as a tidemark region and uncalcified cartilage. CC was found to be more mineralized and the mineral was more crystalline compared with SBP. Both tissues exhibited similar compositional changes as a function of histopathological OA severity. In early OA, the mineralization tends to increase, and the mineral contains fewer carbonate substitutions. Compared with early OA, mineral crystals are rich in carbonate while the overall mineralization decreases in advanced OA. This Raman spectroscopic study advances the methodology for investigating the complex osteochondral junction from native tissue. The developed methodology can be used to elucidate detailed tissue-specific changes in the chemical composition with advancing OA. Statement of SignificanceIn this study, Raman microspectroscopy was utilized to investigate the influence of osteoarthritic degeneration on the tissue-specific biochemical composition of the human osteochondral junction. Multivariate cluster analyses allowed us to characterize subtle compositional changes in the calcified cartilage and subchondral bone plate as well as in the tidemark region. The compositional differences found between the calcified cartilage and subchondral bone plate in both organic and mineral phases will serve as critical benchmark parameters when designing biomaterials for osteochondral repair. We found tissue-specific changes in the mineralization and carbonate substitution as a function of histopathological OA severity. Our developed methodology can be used to investigate the metabolic changes in the osteochondral junction associated with osteoarthritis.

Sample preparation for Raman microspectroscopy

Abstract Raman spectroscopy and its variants allow for the investigation of a wide range of biological and biomedical samples, i. e. tissue sections, single cells and small molecules. The obtained information is on a molecular level. By making use of databases and chemometrical approaches, the chemical composition of complex samples can also be defined. The measurement procedure is straight forward, however most often sample preparation protocols must be implemented. While pure samples, such as high purity powders or highly concentrated chemicals in aqueous solutions, can be directly measured without any prior sample purification step, samples of biological origin, such as tissue sections, pathogens in suspension or biofluids, food and beverages often require pre-processing steps prior to Raman measurements. In this book chapter, different strategies for handling and processing various sample matrices for a subsequent Raman microspectroscopic analysis were introduced illustrating the high potential of this promising technique for life science and medical applications. The presented methods range from standalone techniques, such as filtration, centrifugation or immunocapture to innovative platform approaches which will be exemplary addressed. Therefore, the reader will be introduced to methods that will simplify the complexity of the matrix in which the targeted molecular species are present allowing direct Raman measurements with bench top or portable setups.

DNA-Related Modifications in a Mixture of Human Lympho-Monocyte Exposed to Radiofrequency Fields and Detected by Raman Microspectroscopy Analysis

Human exposure to electromagnetic fields (EMFs) has risen considerably during the last decades, because of the industrial and technical development and the consequent increase of artificial EMFs sources. In particular, blood is largely involved in the environmental EMF exposure, because it is located everywhere in the human body. Lympho-monocyte cells are blood components that protect the human organism against infections. In this study, we investigate biochemical changes in lympho-monocyte cells extracted from human peripheral blood after exposure to EMFs at 1.8 GHz frequency and 200 V/m electric field strength for times ranging from 5 to 20 h inside a reverberation chamber. Some mixtures of cells, coming from many human subjects, were exposed and successively investigated by means of Raman micro-spectroscopy technique and principal components analysis. The spectral analysis was able to detect variations of the biochemical composition of the nucleus of exposed cells. Such modifications are mainly detectable as an intensity decrease of some DNA and nucleic acid Raman peaks with respect to the intensity of some protein peaks and they were most evident in the case of 20 h exposed samples. These results were in agreement with the increase of reactive oxygen species (ROS) production, observed in the exposed cells. Overall, the obtained results point out that EMFs exposure may induce modifications of the DNA in some blood cells of long-term exposed people.

Alpha-keratin and corneous beta protein in the parakeratinized epithelium of the tongue in the domestic goose (Anser anser f. domestica).

The parakeratinized epithelium is a common epithelium in the oral cavity in birds and is characterized by the presence of cell nuclei in the cells of the cornified layer. This epithelium covers almost the entire dorsal surface of the tongue in the domestic goose apart of the lingual nail and conical papillae. So far no study has identified the molecular proteins alpha-keratin (IF-keratin) and/or corneous beta protein (CBP), which are responsible for keratinization or cornification processes in the parakeratinized epithelium of domestic geese. The study was performed using immunohistochemical (IHC) methods to identify alpha-keratin. The innovative method of Raman microspectroscopy was used to determine the presence of CBP and specify their percentage in epithelial layers of the parakeratinized epithelium. The results revealed that alpha-keratin is present in the whole parakeratinized epithelium. A strong staining reaction was detected in the basal and intermediate layers and a less strong staining reaction in the cornified layer. Raman microspectroscopy analysis confirmed the presence of alpha-keratin and demonstrated that its percentage decreases from the basal layer to the cornified layer. The Raman microspectroscopy technique revealed the occurrence of CBP in the parakeratinized epithelium and demonstrated that the percentage of this protein increases from the basal layer to the cornified layer. Performed analysis determines that parakeratinized epithelium undergoes cornification. However, the lower percentage of CBP in the cornified layer of parakeratinized epithelium than in orthokeratinized epithelium points to the fact that parakeratinized epithelium has a weaker protective function.

Amfibol-gazdag harzburgit xenolit fluidumzárvány vizsgálata a Stájer-medencéből

A Stájer-medence a Kárpát-Pannon régió nyugati szélén helyezkedik el, a Pannon-medence és a Keleti-Alpok közötti átmeneti zónában. A Stájer-medencében plio-pleisztocén alkáli bazaltok mintázták meg a medence alatti litoszféraköpenyt. A köpeny eredetű xenolitok közül ritkaságnak számító, modális metaszomatózison átesett, flogopit- és apatit-tartalmú amfibol-gazdag peridotitok is előkerültek. Jelen munkában egy ilyen, gazdagodott xenolit komplex fluidumzárvány vizsgálatát mutatjuk be. Fluidumzárványokat ortopiroxénben és amfibolban azonosítottunk. Utóbbi ásványban ritkán őrződnek meg köpenyfluidumok zárványai, ezért vizsgálatuk egyedi lehetőséget nyújt a köpenyben vándorló fluidumok megismerésére. Az elsődleges és álmásodlagos zárványok összetételét petrográfiai elemzések után mikrotermometriai és Raman-mikrospektroszkópiai mérések segítségével határoztuk meg.
 Az ortopiroxének zárványainak folyadék fázisában a nagy sűrűségű (~1 g/cm3) CO2 a domináns komponens (>95 mol. %), ami mellett H2O-t (<3,5 mol. %) és N2-t (<0,2 mol. %) is kimutattunk. A fluidumzárványokban azonosított szilárd fázisok leányásványok, magnezitből és anhidritből állnak. Amfibolok zárványaiban szintén CO2 a leggyakoribb komponens (>98 mol. %), ami mellett H2O-t (<1,2 mol. %), N2-t (<0,1 mol. %) és a vízben oldott SO42--t azonosítottunk. A szilárd fázisok különböző karbonátokból (magnezit, Na-tartalmú dawsonit, nahkolit és nátrit) és szulfátokból (anhidrit, Na-tartalmú thenardit-burkeit) álló ásványegyüttest alkotnak. A klór-mentes, Na+ és SO42--tartalmú fluidösszetétel egyedinek tekinthető köpenykörülmények között. A fluidum forrása egy illó-gazdag olvadék lehetett, amelyben az amfibol (valamint alárendelten flogopit és apatit) kristályosodása során feldúsultak az illók (C-O-N-S), Na+, HCO3- és SO42-, amelyek csapdázódtak a keletkező amfibolban.

Ultra-slow diffusion of hexacyanoferrate anions in poly(diallyldimethyl ammonium chloride)-poly(acrylic acid sodium salt) multilayer films

Polyelectrolyte multilayer (PEM) films display either a linear or an exponential increase of their thickness with increasing the number of polycation/polyanion deposition cycles. Each of these growth regimes is further associated with a specific mode of internal PEM charge compensation: either intrinsic in the case of linearly growing films, or extrinsic for exponentially growing films with incorporation of small ions from the neighbouring electrolyte solution. In this report, we investigate by electrochemistry the charge compensation mechanism operational for poly(diallyldimethyl ammonium chloride)-poly(acrylic acid sodium salt) (PDAMAC-PAA)n films recently found to be out-of-equilibrium immediately after the deposition process despite of their exponential growth mode. It is found here that these films are defined by a negative Donnan potential drop (ca. −160 mV), thus evidencing an extrinsic charge compensation process, and most importantly, by a remarkably slow kinetics for loading of hexacyanoferrate anions, a very unusual property for PEMs with exponential growth. Depending on the film thickness, the diffusion coefficient of the redox probe in the film is found to be of the order of 10−13 − 10−15 cm2 s−1, i.e. about 8 to 10 orders of magnitude less than that typically measured in aqueous solution. This results in a steady state filling of e.g. a 2.8 µm thick PEM film with the redox probe that is achieved after more than 40 h, a feature in line with the typical 4 to 5 days relaxation timescale of the film structure previously determined by atomic force microscopy and Raman micro-spectroscopy analyses.

The Impact of Antioxidants from the Diet on Breast Cancer Cells Monitored by Raman Microspectroscopy

Background:The impact of the ubiquitous dietary phenolic compound p-coumaric acid on human breast cancer cells was assessed, through a multidisciplinary approach: Combined biological assays for cytotoxicity evaluation and biochemical profiling by Raman microspectroscopic analysis in cells. </P><P> Methods: Para-coumaric acid was shown to exert in vitro chemoprotective and antitumor activities, depending on the concentration and cell line probed: a significant anti-invasive ability was detected for the triple-negative MDA-MB-231 cells, while a high pro-oxidant effect was found for the estrogen- dependent MCF-7 cells. A striking cell selectivity was obtained, with a more noticeable outcome on the triple-negative MDA-MB-231 cell line.Results:The main impact on the cellular biochemical profile was verified to be on proteins and lipids, thus justifying the compound´s anti-invasive effect and chemoprotective ability.Conclusion:p-Coumaric acid was thus shown to be a promising chemoprotective/chemotherapeutic agent, particularly against the low prognosis triple-negative human breast adenocarcinoma.

Resolution of the earliest metazoan record: Differential taphonomy of Ediacaran and Paleozoic fossil molds and casts

Much of our knowledge of early metazoan evolution is derived from unmineralized death mask or endorelief mold and cast fossils in Ediacaran clastic sedimentary rocks. This record is often regarded as a unique ‘Ediacaran taphonomic window’; however, the prevalence of soft-bodied molds and casts in Paleozoic clastic rocks has been increasing, begging an extension, or modification, to our understanding of this preservational motif. Chief amongst such fossils are eldonids, a non-biomineralized group of stem deuterostomes. Because eldonids are also preserved as compressed or flattened fossils from deposits like the Burgess Shale, Chengjiang and Kaili, they offer a comparative case study for evaluating the taphonomic fidelity of mold/cast-style preservation during this interval. EDS and Raman microspectroscopic analysis of Ordovician and Devonian eldonid molds and casts, and comparison with Burgess Shale eldonids, suggests the mold/cast taphonomic style produces significantly lower fidelity of fossil preservation. We propose that eldonid mold/cast fossils are preserved by the adsorption of reduced iron ions onto tissues composed primarily of high molecular weight (HMW) biopolymers which require enzymatic degradation prior to decay. Nucleation and growth of aluminosilicates and/or sulfides around these adsorbed ions forms a fossilizable surface veneer, preserving a death mask mold. More labile tissues could not be fossilized in this mold and cast style. Ediacaran mold and cast fossils from South Australia, the White Sea region of Russia, Namibia, and Newfoundland exhibit preservational characteristics consistent with this new proposed model. Analysis of their preservational mode suggests that the first metazoans, which would have lacked HMW biopolymeric tissues, could not have been fossilized in this particular style. Thus, understanding the origin and earliest evolution of the Metazoa requires a focus on alternative modes of fossilization.

Improved long-term durability of allogeneic heart valves in the orthotopic sheep model.

Frozen cryopreservation (FC) with the vapour phase of liquid nitrogen storage (-135°C) is a standard biobank technique to preserve allogeneic heart valves to enable a preferable allograft valve replacement in clinical settings. However, their long-term function is limited by immune responses, inflammation and structural degeneration. Ice-free cryopreserved (IFC) valves with warmer storage possibilities at -80°C showed better matrix preservation and decreased immunological response in preliminary short-term in vivo studies. Our study aimed to assess the prolonged performance of IFC allografts in an orthotopic pulmonary sheep model. FC (n = 6) and IFC (n = 6) allografts were transplanted into juvenile Merino sheep. After 12 months of implantation, functionality testing via 2-dimensional echocardiography and histological analyses was performed. In addition, multiphoton autofluorescence imaging and Raman microspectroscopy analysis were applied to qualitatively and quantitatively assess the matrix integrity of the leaflets. Six animals from the FC group and 5 animals from the IFC group were included in the analysis. Histological explant analysis showed early inflammation in the FC valves, whereas sustainable, fully functional, devitalized acellular IFC grafts were obtained. IFC valves showed excellent haemodynamic data with fewer gradients, no pulmonary regurgitation, no calcification and acellularity. Structural remodelling of the leaflet matrix structure was only detected in FC-treated tissue, whereas IFC valves maintained matrix integrity comparable to that of native controls. The collagen crimp period and amplitude and elastin structure were significantly different in the FC valve cusps compared to IFC and native cusps. Collagen fibres in the FC valves were less aligned and straightened. IFC heart valves with good haemodynamic function, reduced immunogenicity and preserved matrix structures have the potential to overcome the known limitations of the clinically applied FC valve.

Development of a surface tension mediated technique for dry stabilization of mammalian cells.

Dry state preservation at ambient temperatures (lyopreservation) is a biomimetic alternative to low temperature stabilization (cryopreservation) of biological materials. Lyopreservation is hypothesized to rely upon the creation of a glassy environment, which is commonly observed in desiccation-tolerant organisms. Non-uniformities in dried samples have been indicated as one of the reasons for instability in storage outcome. The current study presents a simple, fast, and uniform surface tension based technique that can be implemented for lyopreservation of mammalian cells. The technique involves withdrawing cells attached to rigid substrates to be submerged in a solution of lyoprotectant and then withdrawing the samples at a specific rate to an inert environment. This creates a uniform thin film of desiccated lyoprotectant due to sudden change of surface tension. The residual moisture contents at different locations in the desiccated film was quantified using a spatially resolved Raman microspectroscopy technique. Post-desiccation cellular viability and growth are quantified using fluorescent microscopy and dye exclusion assays. Cellular injury following desiccation is evaluated by bioenergetic quantification of metabolic functions using extracellular flux analysis and by a Raman microspectroscopic analysis of change in membrane structure. The technique developed here addresses an important bottleneck of lyoprocessing which requires the fast and uniform desiccation of cellular samples.

Raman micro-spectroscopy analysis of human lens epithelial cells exposed to a low-dose-range of ionizing radiation

Recent findings in populations exposed to ionizing radiation (IR) indicate dose-related lens opacification occurs at much lower doses (<2 Gy) than indicated in radiation protection guidelines. As a result, research efforts are now being directed towards identifying early predictors of lens degeneration resulting in cataractogenesis. In this study, Raman micro-spectroscopy was used to investigate the effects of varying doses of radiation, ranging from 0.01 Gy to 5 Gy, on human lens epithelial (HLE) cells which were chemically fixed 24 h post-irradiation. Raman spectra were acquired from the nucleus and cytoplasm of the HLE cells. Spectra were collected from points in a 3 × 3 grid pattern and then averaged. The raw spectra were preprocessed and principal component analysis followed by linear discriminant analysis was used to discriminate between dose and control for 0.25, 0.5, 2, and 5 Gy. Using leave-one-out cross-validation accuracies of greater than 74% were attained for each dose/control combination. The ultra-low doses 0.01 and 0.05 Gy were included in an analysis of band intensities for Raman bands found to be significant in the linear discrimination, and an induced repair model survival curve was fit to a band-difference-ratio plot of this data, suggesting HLE cells undergo a nonlinear response to low-doses of IR. A survival curve was also fit to clonogenic assay data done on the irradiated HLE cells, showing a similar nonlinear response.

Toxicological assessment of nanomaterials: the role of in vitro Raman microspectroscopic analysis.

The acceleration of nanomaterials research has brought about increased demands for rapid analysis of their bioactivity, in a multi-parametric fashion, to minimize the gap between potential applications and knowledge of their toxicological properties. The potential of Raman microspectroscopy for the analysis of biological systems with the aid of multivariate analysis techniques has been demonstrated. In this study, an overview of recent efforts towards establishing a 'label-free high content nanotoxicological assessment technique' using Raman microspectroscopy is presented. The current state of the art for cellular toxicity assessment and the potential of Raman microspectroscopy are discussed, and the spectral markers of the cellular toxic responses upon exposure to nanoparticles, changes on the identified spectral markers upon exposure to different nanoparticles, cell death mechanisms, and the effects of nanoparticles on different cell lines are summarized. Moreover, 3D toxicity plots of spectral markers, as a function of time and dose, are introduced as new methodology for toxicological analysis based on the intrinsic properties of the biomolecular changes, such as cytoplasmic RNA aberrations, protein and lipid damage associated with the toxic response. The 3D evolution of the spectral markers are correlated with the results obtained by commonly used cytotoxicity assays, and significant similarities are observed between band intensity and percentage viability obtained by the Alamar Blue assay, as an example. Therefore, the developed 3D plots can be used to identify toxicological properties of a nanomaterial and can potentially be used to predict toxicity, which can provide rapid advances in nanomedicine. Graphical Abstract Spectral markers of cytotoxicity as a function of time and dose.

Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells.

Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ∼260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation.

Localization of Alpha-Keratin and Beta-Keratin (Corneous Beta Protein) in the Epithelium on the Ventral Surface of the Lingual Apex and Its Lingual Nail in the Domestic Goose (Anser Anser f. domestica) by Using Immunohistochemistry and Raman Microspectroscopy Analysis.

The epithelium of the ventral surface of the apex of the tongue in most birds is specified by the presence of the special superficial layer called lingual nail. The aim of the present study is to determine the localization of the alpha-keratin and beta-keratin (corneous beta protein) in this special epithelium in the domestic goose by using immunohistochemistry staining and the Raman spectroscopy analysis. Due to lack of commercially available antibodies to detect beta-keratin (corneous beta protein), the Raman spectroscopy was used as a specific tool to detect and describe the secondary structure of proteins. The immunohistochemical (IHC) detections reveal the presence of alpha-keratin in all layers of the epithelium, but significant differences in the distribution of the alpha-keratin in the epithelial layers appear. The staining reaction is stronger from the basal layer to the upper zone of the intermediate layer. The unique result is weak staining for the alpha-keratin in the lingual nail. Applications of the Raman spectroscopy as a complementary method not only confirmed results of IHC staining for alpha-keratin, but showed that this technique could be used to demonstrate the presence of beta-keratin (corneous beta protein). Functionally, the localization of alpha-keratin in the epithelium of the ventral surface of the lingual apex provides a proper scaffold for epithelial cells and promotes structural integrity, whereas the presence of beta-keratin (corneous beta protein) in the lingual nail, described also as exoskeleton of the ventral surface of the apex, endures mechanical stress. Anat Rec, 300:1361-1368, 2017. © 2017 Wiley Periodicals, Inc.

Raman micro-spectroscopy analysis of different sperm regions: a species comparison.

Is Raman micro-spectroscopy a valid approach to assess the biochemical hallmarks of sperm regions (head, midpiece and tail) in four different species? Non-invasive Raman micro-spectroscopy provides spectral patterns enabling the biochemical characterization of the three sperm regions in the four species, revealing however high similarities for each region among species. Raman micro-spectroscopy has been described as an innovative method to assess sperm features having the potential to be used as a non-invasive selection tool. However, except for nuclear DNA, the identification and assignment of spectral bands in Raman-profiles to the different sperm regions is scarce and controversial. Raman spectra from head, midpiece and tail of four different species were obtained. Sperm samples were collected and smeared on microscope slides. Air dried samples were subjected to Raman analysis using previously standardized procedures. Sperm samples from (i) two donors attending the infertility clinic at the Centre of Reproductive Medicine and Andrology; (ii) two C57BL/6 -TgN (ACTbEGFP) 1Osb adult mice; (iii) two adult Cynomolgus monkeys (Macaca fascicularis) and (iv) two sea urchins (Arbacia punctulata) were used to characterize and compare their spectral profiles. Differences and similarities were confirmed by principal component analysis (PCA). Several novel region-specific peaks were identified. The three regions could be differentiated by distinctive Raman patterns irrespective of the species. However, regardless of the specie, their main spectral pattern remains mostly unchanged. These results were corroborated by the PCA analysis and suggest that the basic constituents of spermatozoa are biochemically similar among species. Further research should be performed in live sperm to validate the detected spectral bands and their use as markers of distinctive regions. Raman peaks that have never been described in the sperm cell were detected. Particularly important are those that are unique to the midpiece as they might be a reference to the identification of sperm mitochondria, whose function is highly correlated with that of sperm. In the future, Raman micro-spectroscopy has the potential to be applied in assessment of male fertility. N/A. This work was supported by BMBF project 'Sperm Ident' (FKZ:13N13024) and the DAAD-CRUP bilateral exchange program (AI A06/16-57213087). S.A. is a recipient of a fellowship from the Portuguese foundation for science and technology (FCT-SFRH/BPD/110160/2015) and R.DC. is a recipient of a DAAD PhD stipend (91590556). There is no competing interest.

Development of methodology for Raman microspectroscopic analysis of oral exfoliated cells

A standardised protocol for sample collection, sample preparation, spectral acquisition and data processing for future studies of oral exfoliated cells based on Raman microspectroscopy.