Raman Microspectroscopy Research Articles

Human glabrous skin contains crystallized urea dendriform structures in the stratum corneum which affect the hydration levels.

Glabrous skin is hair-free skin with a high density of sweat glands, which is found on the palms, and soles of mammalians, covered with a thick stratum corneum. Dry hands are often an occupational problem which deserves attention from dermatologists. Urea is found in the skin as a component of the natural moisturizing factor and of sweat. We report the discovery of dendrimer structures of crystalized urea in the stratum corneum of palmar glabrous skin using laser scanning microscopy. The chemical and structural nature of the urea crystallites was investigated in vivo by non-invasive techniques. The relation of crystallization to skin hydration was explored. We analysed the index finger, small finger and tenar palmar area of 18 study participants using non-invasive optical methods, such as laser scanning microscopy, Raman microspectroscopy and two-photon tomography. Skin hydration was measured using corneometry. Crystalline urea structures were found in the stratum corneum of about two-thirds of the participants. Participants with a higher density of crystallized urea structures exhibited a lower skin hydration. The chemical nature and the crystalline structure of the urea were confirmed by Raman microspectroscopy and by second harmonic generated signals in two-photon tomography. The presence of urea dendrimer crystals in the glabrous skin seems to reduce the water binding capacity leading to dry hands. These findings highlight a new direction in understanding the mechanisms leading to dry hands and open opportunities for the development of better moisturizers and hand disinfection products and for diagnostic of dry skin.

The recovery of aerosol-sized microplastics in highly refractory vegetal matrices for identification by automated Raman microspectroscopy

Ombrotrophic peatlands are fed uniquely by atmospheric inputs and therefore have much potential as temporal archives of atmospheric microplastic (MP) deposition, yet the recovery and detection of MP within an almost purely organic matrix is challenging. This study presents a novel peat digestion protocol using sodium hypochlorite (NaClO) as a reagent for biogenic matrix removal. NaClO is more efficient than hydrogen peroxide (H2O2). By using purged air-assisted digestion, NaClO (50 vol%) reached 99% matrix digestion compared with 28% and 75% by H2O2 (30 vol%) and Fenton's reagent, respectively. At a concentration of 50 vol% NaClO did however chemically disintegrate small amounts (<10 mass %) of polyethylene terephthalate (PET) and polyamide (PA) fragments in the millimeter size range. Observation of PA6 in natural peat samples, while not found in the procedural blanks, questions whether PA is fully disintegrated by NaClO. The protocol was applied to three commercial sphagnum moss test samples, in which MP particles in the range of 0.8–65.4 μm were detected by Raman microspectroscopy. The MP mass% was determined at 0.012% corresponding to 129 thousand MP particles/g, of which 62% were smaller than 5 μm and 80% were smaller than 10 μm, yet were accountable for only 0.4% (500 ng) and 3.2% (4 μg) of the total mass of MP, respectively. These findings underline the importance of the identification of particles Ø < 5 μm when investigating atmospheric MP deposition. The MP counts were corrected for MP recovery loss and procedural blank contamination. MP spike recovery following the full protocol was estimated at 60%. The protocol offers an efficient way of isolating and pre-concentrating most aerosol sized MPs in large quantities of refractory vegetal matrices and enables the automated μRaman scanning of thousands of particles at a spatial resolution on the order of 1 μm.

Graphitic inclusions in zircon from early Phanerozoic S-type granite: Implications for the preservation of Hadean biosignatures

A biotic origin of isotopically light graphite in Hadean zircon remains contested, in part because it is unclear how biogenic carbon in sediments behaves during diagenesis, metamorphism and anatexis, and how it can be preserved as inclusions in zircon. Here, we report the discovery of graphitic inclusions in zircon from Rumburk granite in the Lusatian Block of eastern central Europe as the first example of such inclusion-bearing zircon in a well-constrained geological and petrological context. Most graphite inclusions are trapped at the interface between inherited zircon interiors of early Cambrian–Proterozoic age and late Cambrian (496.2 ± 2.3 Ma; 95% confidence) zircon overgrowths that crystallized at ∼700 °C under highly reducing conditions (typically −4.6 log units relative to the fayalite-magnetite-quartz buffer). Zircon overgrowths are also enriched in xenotime component (up to 93 µmol/g P) and δ18O (average δ18O = 7.7‰), both typical for S-type granitic melts. Raman microspectroscopy reveals crystalline and disordered graphitic carbon, but because overgrowth temperatures reached >700 °C, sufficient for complete graphitization, disordering is presumably secondary and caused by in-situ irradiation from the zircon host. Because organic materials were avoided during sample preparation, and inclusions were excavated by ion beam sputtering, exposure to potential C-bearing contaminants can be dismissed. Inclusions range in δ13C from −44.6 to −7.5‰ with a dominant mode at δ13C = −34‰ that is positively skewed. Rayleigh-type graphite precipitation from a CO2-CH4 fluid with a starting composition equivalent to regional black shale (δ13C = −32‰) explains inclusion carbon isotopic range and distribution. Collectively, these observations suggest that graphite was initially trapped in voids formed via dissolution-reprecipitation of trace element enriched, and possibly metamict, detrital zircon in metasedimentary protoliths when exposed to C-O-H fluids during prograde metamorphism. Subsequently, graphite-filled voids in inherited zircon became enclosed when zircon rims crystallized from highly reduced S-type granitic melts. Besides partial disordering of graphitic carbon due to irradiation, there is no indication for post-entrapment alteration, demonstrating that graphitic inclusions in zircon can preserve isotopic biosignatures over hundreds of millions of years.

Critical assessment of approach towards estimation of microplastics in environmental matrices

AbstractMicroplastics (MPs) are the contaminants of growing concern due to their persistent properties in the environment. This review compares the global data on MPs concentration in soil, sediment, salt, sand, biosolids, and water. Previous studies have also developed various methods to estimate MPs for different environmental matrices, which were generally based on salt‐based density separation, physical (microscopy), chemical characterization (spectroscopy), and visual counting for extraction, identification, and quantification, respectively. In most of the existing field‐based studies, MPs are usually identified and detected by microscopic observation and Fourier transform infrared (FTIR) analysis, which maximize the probability of error in the estimation process. A combination of thermogravimetric analysis‐FTIR spectroscopy, Raman microspectroscopy, optical photothermal infrared, and pyrolysis/gas chromatography–mass spectrometry are used for the identification, recognition, and quantification of MPs in the heterogeneous solid and liquid matrices for minimizing the interference and misinterpretation of the other constituents (organic matter) present in the samples, especially soil, sediments, and biosolids.

A Study on Manufacturing Technique and Corrosion Characteristics of Bronze Dirk, Bronze Sword, Bronze Mirror in Early Iron Age Excavated from the Osong Site in Cheongju

Metallography, SEM-EDS, and Raman Micro-Spectroscopy analysis were conducted on bronze dirk, bronze sword, and bronze mirror excavated from the pit tomb No. 1-1 at 12th Point of Osong Site in Cheongju to study the manufacturing techniques and corrosion properties. The bronze dirk, bronze sword and bronze mirror are judged to be binary alloys of Cu(79.16∼79.89 wt%)-Sn (19.12∼20.34 wt%). The microstructure is a cast structure composed of δ phase and (α+δ) eutectoid, and no heat treatment was performed. As a result of classifying the corrosion characteristics, it was confirmed that the bronze dirk, bronze sword, bronze mirror were Type I. As a result of EDS analysis in subsurface area, tin oxide is located as a selective corrosion of (α+δ) eutectoid, and as a result of Raman Micro-Spectroscopy analysis, corrosion products of Cassiterite and Malachite were identified for the bronze dirk and Cassiterite for bronze sword and bronze mirror. The secondary metallic copper was mainly present in the hole left by corrosion of Pb particles and on the (α+δ) eutectoid, and was found to be 100.00 wt% of Cu as a result of the analysis, confirming that all of them were high-purity copper.

Simultaneous Imaging and Characterization of Polyunsaturated Fatty Acids, Carotenoids, and Microcrystalline Guanine in Single Aurantiochytrium limacinum Cells with Linear and Nonlinear Raman Microspectroscopy.

Thraustochytrids are heterotrophic marine protists known for their high production capacity of various compounds with health benefits, such as polyunsaturated fatty acids and carotenoids. Although much effort has been focused on developing optimal cultivation methods for efficient microbial production, these high-value compounds and their interrelationships are not well understood at the single-cell level. Here we used spontaneous (linear) Raman and multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy to visualize and characterize lipids (e.g., docosahexaenoic acid) and carotenoids (e.g., astaxanthin) accumulated in single living Aurantiochytrium limacinum cells. Spontaneous Raman imaging with the help of multivariate curve resolution-alternating least-squares enabled us to make unambiguous assignments of the molecular components we detected and derive their intracellular distributions separately. Near-IR excited CARS imaging yielded the Raman images at least an order of magnitude faster than spontaneous Raman imaging, with suppressed contributions of carotenoids. As the culture time increased from 2 to 5 days, the lipid amount increased by a factor of ∼7, whereas the carotenoid amount did not change significantly. Furthermore, we observed a highly localized component in A. limacinum cells. This component was found to be mixed crystals of guanine and other purine derivatives. The present study demonstrates the potential of the linear-nonlinear Raman hybrid approach that allows for accurate molecular identification and fast imaging in a label-free manner to link information derived from single cells with strategies for mass culture of useful thraustochytrids.

Thermoluminescent characterization and defect studies of graphite-rich media under high dose neutron exposure

Thermoluminescence (TL) materials have a broad variety of uses in various fields, such as clinical research, individual dosimetry, and environmental dosimetry, amongst others. However, the use of individual neutron dosimetry has been developing more aggressively lately. In this regard, present study establishes a relationship between the neutron dosage and the optical property changes of graphite-rich materials caused by high doses of neutron radiation. This has been done with the intention of developing a novel, graphite-based radiation dosimeter. Herein, the TL yield of commercially graphite-rich materials (i.e. graphite sheet, 2B and HB grade pencils) irradiated by neutron radiation with doses ranging from 250 Gy to 1500 Gy has been investigated. The samples were bombarded with thermal neutrons as well as a negligible amount of gamma rays, from the nuclear reactor TRIGA-II installed at the Bangladesh Atomic Energy Commission. The shape of the glow curves was observed to be independent of the given dosage, with the predominant TL dosimetric peak maintained within the region of 163 °C–168 °C for each sample. By studying the glow curves of the irradiated samples, some of the most well theoretical models and techniques were used to compute the kinetic parameters such as the order of kinetics (b), activation energy (E) or trap depth, frequency factor (s) or escape probability, and trap lifetime (τ). All of the samples were found to have a good linear response over the whole dosage range, with 2B grade of polymer pencil lead graphite (PPLGs) demonstrating a higher level of sensitivity than both HB grade and graphite sheet (GS) samples. Additionally, the level of sensitivity shown by each of them is highest at the lowest dosage that was given, and it decreases as the dose increases. Importantly, the phenomenon of dose-dependent structural modifications and internal annealing of defects has been observed by assessing the area of deconvoluted micro-Raman spectra of graphite-rich materials in high-frequency areas. This trend is consistent with the cyclical pattern reported in the intensity ratio of defect and graphite modes in previously investigated carbon-rich media. Such recurrent occurrences suggest the idea of employing Raman microspectroscopy as a radiation damage study tool for carbonaceous materials. The excellent responses of the key TL properties of the 2B grade pencil demonstrate its usefulness as a passive radiation dosimeter. As a consequence, the findings suggest that graphite-rich materials have the potential to be useful as a low-cost passive radiation dosimeter, with applications in radiotherapy and manufacturing.

Deuterium isotope probing (DIP) on Listeria innocua: Optimisation of labelling and impact on viability state.

An innovative approach, Raman microspectroscopy coupled with deuterium isotope probing (Raman-DIP), can be used to evaluate the metabolism of deuterated carbon source in bacteria and also to presume different anabolic pathways. This method requires the treatment of cells with heavy water that could affect the bacterial viability state at higher concentration. In this study, we evaluated the effect of heavy water incorporation on the viability state of Listeria innocua cells. We exposed the L. innocua suspensions to different heavy water concentrations (0%, 25%, 50% and 75%) from 30 minutes to 72 h of incubation times at 37°C. The total, viable and viable culturable populations were quantified by qPCR, PMA-qPCR and plate count agar respectively. We analyzed heavy water incorporation by Raman-DIP. The exposure of L. innocua cells to different concentrations of heavy water did not alter their cell viability to 24 h incubation time. In addition, the maximum intensity for C-D band, specific for the incorporation of heavy water, was reached after 2 h of exposure in a media containing 75% v/v D2O but an early detection of the labelling was possible at t = 1 h 30 min. In conclusion, the use of D2O as a metabolic marker was validated and can be developed for the detection of L. innocua cell viability state.

Study of the oxidation process of bismuth nanoparticles using NaClO

Bismuth nanoparticles (NPs) colloids synthesized in deionized water by laser ablation of solids in liquids technique (LASL) were oxidized using NaClO solutions at different concentrations. Oxidized nanomaterials were characterized using several techniques. The crystalline phases of the bismuth compound were determined using Raman microspectroscopy, and the crystallographic structure was identified by x-ray diffraction (XRD). The size and morphology of the obtained nanomaterials were studied by transmission electron microscopy (TEM). The chemical states were determined using x-ray photoelectron spectroscopy (XPS), and the optical properties of the colloids were characterized by UV–vis spectroscopy. The absorption spectra were analyzed using the Tauc method to determine the band gaps of the obtained nanomaterials. Our results showed morphological changes, starting from small nanoparticles to nanosheets and a mixture of nanosheets with hollow nanoparticles. Two kinds of nanomaterials were found depending on the NaClO solution concentration: Bi2O2CO3 single phase and a mixture of δ-Bi2O3 and Bi2O2CO3. Some samples were tested as photocatalysts and showed good performance in the degradation of methylene blue in solution. To the best of our knowledge, this is the first study on the oxidation process of bismuth colloidal nanoparticles at room temperature.

Multiple roads lead to Rome: unique morphology and chemistry of endospores, exospores, myxospores, cysts and akinetes in bacteria.

The production of specialized resting cells is a remarkable survival strategy developed by many organisms to withstand unfavourable environmental factors such as nutrient depletion or other changes in abiotic and/or biotic conditions. Five bacterial taxa are recognized to form specialized resting cells: Firmicutes, forming endospores; Actinobacteria, forming exospores; Cyanobacteria, forming akinetes; the δ-Proteobacterial order Myxococcales, forming myxospores; and Azotobacteraceae, forming cysts. All these specialized resting cells are characterized by low-to-absent metabolic activity and higher resistance to environmental stress (desiccation, heat, starvation, etc.) when compared to vegetative cells. Given their similarity in function, we tested the potential existence of a universal morpho-chemical marker for identifying these specialized resting cells. After the production of endospores, exospores, akinetes and cysts in model organisms, we performed the first cross-species morphological and chemical comparison of bacterial sporulation. Cryo-electron microscopy of vitreous sections (CEMOVIS) was used to describe near-native morphology of the resting cells in comparison to the morphology of their respective vegetative cells. Resting cells shared a thicker cell envelope as their only common morphological feature. The chemical composition of the different specialized resting cells at the single-cell level was investigated using confocal Raman microspectroscopy. Our results show that the different specialized cells do not share a common chemical signature, but rather each group has a unique signature with a variable conservation of the signature of the vegetative cells. Additionally, we present the validation of Raman signatures associated with calcium dipicolinic acid (CaDPA) and their variation across individual cells to develop specific sorting thresholds for the isolation of endospores. This provides a proof of concept of the feasibility of isolating bacterial spores using a Raman-activated cell-sorting platform. This cross-species comparison and the current knowledge of genetic pathways inducing the formation of the resting cells highlights the complexity of this convergent evolutionary strategy promoting bacterial survival.

Resonance Raman and Visible Micro-Spectroscopy for the In-Vivo and In-Vitro Characterization of Anthocyanin-Based Pigments in Blue and Violet Flowers: A Comparison with HPLC-ESI- MS Analysis of the Extracts

Microanalysis techniques based on resonance Raman and reflection visible spectroscopy have been applied to the characterization of pigments responsible for the blue or violet coloration in flowers; in particular of Lobelia erinus, Campanula portenschlagiana, Cineraria, Viola tricolor, Anemone coronaria, Agapanthus, Platycodon, Salvia farinacea, Plumbago capensis, Ceratostigma plumbaginoides, Commelina communis and Salvia patens. The spectroscopic methods were applied both in vivo on the flower petals and in vitro on extracts obtained through a procedure based on SPE (solid-phase extraction) optimized for minimal quantities of vegetable raw material. Different patterns obtained for the Raman spectra have been correlated, also on the basis of density functional theory (DFT) calculations, with different schemes of substitution of the benzopyrilium nucleus of the anthocyanins and with various possible forms of copigmentation responsible for the stabilization of the blue color. The results obtained were verified by comparison with the analysis of the extracts by HPLC-ESI-MS (liquid chromatography-electrospray ionization-mass spectrometry).

Enamel Structure Defects in Kdf1 Missense Mutation Knock-in Mice.

The Keratinocyte differentiation factor 1 (KDF1) is reported to take part in tooth formation in humans, but the dental phenotype of Kdf1 mutant mice has not been understood. Additionally, the role of the KDF1 gene in dental hard tissue development is rarely known. In this study, we constructed a Kdf1 missense mutation knock-in mouse model through CRISPR/Cas9 gene-editing technology. Enamel samples from wildtypes (WT) and Kdf1 homozygous mutants (HO) were examined using micro-computed tomography (micro-CT), scanning electron microscopy (SEM), an atomic force microscope (AFM) and Raman microspectroscopy. The results showed that a novel Kdf1 missense mutation (c. 908G>C, p.R303P) knock-in mice model was constructed successfully. The enamel of HO mice incisors appeared chalky and defective, exposing the rough interior of the inner enamel and dentin. Micro-CT showed that HO mice had lower volume and mineral density in their tooth enamel. In addition, declined thickness was found in the unerupted enamel layer of incisors in the HO mice. Using SEM and AFM, it was found that enamel prisms in HO mice enamel were abnormally and variously shaped with loose decussating crystal arrangement, meanwhile the enamel rods were partially fused and collapsed, accompanied by large gaps. Furthermore, misshapen nanofibrous apatites were disorderly combined with each other. Raman microspectroscopy revealed a compromised degree of order within the crystals in the enamel after the Kdf1 mutation. To conclude, we identified enamel structure defects in the Kdf1 missense mutation knock-in mice, which displayed fragmentary appearance, abnormally shaped prism structure, decreased mineral density, altered crystal ordering degree and chemical composition of the enamel layer. This may support the potential role of the KDF1 gene in the natural development of enamel.

Quantified analysis of 2D dispersion of carbon nanotubes in hardened cement composite using confocal Raman microspectroscopy

Dispersion of carbon nanotubes (CNTs) is the critical factor affecting the performance of nanocomposites; however, the quality of CNT dispersion could be indirectly understood due to the limitations of evaluation methods of the dispersion. This study aimed to visualize the macroscopic spatial distribution of CNTs in an as-is hardened cement matrix for the quantification of dispersion. Using a confocal Raman microspectroscopy equipped with an optical microscope, the major phases in cement, hydrates, and CNTs were identified from their characteristic Raman signals to construct the definitive phase map. The dispersion efficiency and heterogeneity were defined to quantify the fraction of CNT-affected region and the unevenness of CNT distribution. The experimental results demonstrate the importance of a well-dispersed stable state in CNT suspension and suggest that it is possible to evaluate the effectiveness of ultrasonication with superplasticizers that have been frequently adopted as a dispersion technique in hardened cement composites.

Non-Invasive On-Site pXRF Analysis of Coloring Agents, Marks and Enamels of Qing Imperial and Non-Imperial Porcelain

On-site pXRF analysis in various French collections (Musée du Louvre, Musée national des Arts asiatiques-Guimet, Paris) of porcelains decorated with painted enamels from the Qing Dynasty, in particular porcelains bearing an imperial mark, identifies the types of enamels/glazes, the ions and coloring phases or the opacifier. The study of the elements associated with cobalt (nickel, manganese, arsenic, etc.) and of the impurities of the silicate matrix (yttrium, rubidium and strontium) differentiates the use of ‘Chinese/Asian’ raw materials from ones imported from Europe by the initiative of the European missionaries (chiefly Jesuits) present at the Court (Beijing). Particular attention is paid to the analysis of the blue color of the marks and to the elements associated with the use of gold or copper nanoparticles as well as to the compositions of the pyrochlore phases (tin yellow, Naples yellow). The comparison is extended to pXRF and Raman microspectroscopy measurements previously made on other Qing imperial porcelains as well as on Cantonese productions (on porcelain or metal) from different Swiss and French museums and blue-and-white wares of the Ming and Yuan Dynasties (archaeological and private collections).

In vitro Raman imaging of human macrophages: Impact of eicosapentaenoic acid on the hydrolysis of cholesterol esters in lipid droplets

Atherosclerosis – a cardiovascular disease and the primary cause of morbidity and mortality in industrialized countries – is linked to the existence of atherosclerotic plaques characterized by cholesterol-laden macrophages called foam cells. In these cells, cholesterol esters associated with triglycerides form lipid droplets (LD). The only way to remove this excess cholesterol is to promote free cholesterol efflux from macrophages to specific acceptors. It has been shown recently that eicosapentaenoic acid (EPA) reduces efflux on cholesterol-loaded THP-1 macrophages in vitro due to decreased cholesterol esters hydrolysis. These in vitro observations could reflect EPA's difficulty in facilitating in vivo the antiatherogenic process of cholesterol efflux within advanced atherosclerotic plaques.This work aims to study in vitro the impact of EPA on cholesterol esters hydrolysis in the LD of human THP-1 macrophages using vibrational Raman microspectroscopy. For this, we used deuterated EPA and recorded spectral images at the cell scale after different hydrolysis times. Resultsshowed that EPA is involved in forming triglycerides and phospholipids of LD. Hydrolysis kinetics slowed down after 24 h, triglycerides increased, and the intensity of the characteristic bands linked to deuteration decreased. The size of LD without hydrolysis (H0) is higher than that after 24 h (H1) or 48 h (H2) of hydrolysis. The size decrease is sharper when going from H0 to H1 than from H1 to H2. Principal component analysis illustrated data’ projection according to the cellular compartment, the hydrolysis time, and the supplementation of the medium.

Baseline assessment of microplastics in commercially important marine bivalves from New York, U.S.A.

Microplastic (MP) contamination in bivalve mollusks has become a significant concern over the last few years. These ecologically and economically valuable species are popular seafood items for human consumption. As filter feeders, bivalves may ingest MPs in their bodies, possibly impacting their physiology and fitness. Additionally, a considerable amount of the seafood that humans consume comes from coastal areas where MP concentrations tend to be the highest. This research provides the first examination of MPs in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) that were grown locally in coastal areas of New York, contributing to a baseline for the northeast and mid-Atlantic regions of the U.S. A total of 48 eastern oysters (n = 12 per site, at four sites) and hard clams (n = 24 per site, at two sites) were sampled in summer 2021. While MP fibers and fragments (i.e. polyethylene terephthalate, polystyrene, and polypropylene) were found in some oysters, other contaminants (e.g. indigo dye, phthalocyanine, dye 823, etc.) were found in both bivalve species. Particle composition was verified using Raman microspectroscopy. Although mean MP concentrations were low in eastern oysters (i.e. 0.008 MPs g−1 of soft tissue wet weight; 0.125 MPs ind−1) and not found in hard clams, more research is needed to assess the magnitude of contamination in these edible bivalves.

Brillouin–Raman micro-spectroscopy and machine learning techniques to classify osteoarthritic lesions in the human articular cartilage

In this study, Brillouin and Raman micro-Spectroscopy (BRamS) and Machine Learning were used to set-up a new diagnostic tool for Osteoarthritis (OA), potentially extendible to other musculoskeletal diseases. OA is a degenerative pathology, causing the onset of chronic pain due to cartilage disruption. Despite this, it is often diagnosed late and the radiological assessment during the routine examination may fail to recognize the threshold beyond which pharmacological treatment is no longer sufficient and prosthetic replacement is required. Here, femoral head resections of OA-affected patients were analyzed by BRamS, looking for distinctive mechanical and chemical markers of the progressive degeneration degree, and the result was compared to standard assignment via histological staining. The procedure was optimized for diagnostic prediction by using a machine learning algorithm and reducing the time required for measurements, paving the way for possible future in vivo characterization of the articular surface through endoscopic probes during arthroscopy.

Pd-Based Hybrid Nanoparticles As Multimodal Theranostic Nanomedicine.

A nanodelivery system based on palladium nanoparticles (PdNP) and cisplatin (CisPt) was developed by physisorption of the drug onto the PdNP synthesized via a green redox process, using d-glucose and polyvinylpyrrolidone (PVP) as reducing and stabilizing/capping agents, respectively. UV-vis analysis and H2-evolution measurements were carried out to prove the nanoparticles' capability to act as bimodal theranostic nanomedicine, i.e., having both plasmonic and photocatalytic properties. XPS, XRD, and TEM allowed light to be shed on the chemical composition and morphology of the PdNP. The analysis of the UV-visible spectra evidenced plasmonic peak changes for the hybrid nanoparticle-drug assembly (Pd@CisPt), which pointed to a significant interaction of CisPt with the NP surface. The drug loading was quantitatively estimated by ICP-OES measurements, while DLS and AFM confirmed the strong association of the drug with the nanoparticle surface. The test of SOD-like activity in a cell-free environment proved the maintenance of the antioxidant capability of PdNP also in the Pd@CisPt systems. Finally, Pd@CisPt tested in prostate cancer cells (PC-3 line) unveiled the antitumoral action of the developed nanomedicine, related to reactive oxygen species (ROS) generation, with a condition of protein misfolding/unfolding and DNA damage, as evidenced by cytotoxicity and MitoSOX assays, as well as Raman microspectroscopy, respectively. Cell imaging by confocal microscopy evidenced cellular uptake of the nanoparticles, as well as dynamic processes of copper ion accumulation at the level of subcellular compartments. Finally, cell migration studies upon treatment with Pd@CisPt evidenced a tunable response between the inhibitory effect of CisPt and the enhanced rate of cell migration for the metal NP alone, which pointed out the promising potential of the developed theranostic nanomedicine in tissue regeneration.

Understanding Raman Spectral Based Classifications with Convolutional Neural Networks Using Practical Examples of Fungal Spores and Carotenoid-Pigmented Microorganisms

Numerous publications showing that robust prediction models for microorganisms based on Raman micro-spectroscopy in combination with chemometric methods are feasible, often with very precise predictions. Advances in machine learning and easier accessibility to software make it increasingly easy for users to generate predictive models from complex data. However, the question regarding why those predictions are so accurate receives much less attention. In our work, we use Raman spectroscopic data of fungal spores and carotenoid-containing microorganisms to show that it is often not the position of the peaks or the subtle differences in the band ratios of the spectra, due to small differences in the chemical composition of the organisms, that allow accurate classification. Rather, it can be characteristic effects on the baselines of Raman spectra in biochemically similar microorganisms that can be enhanced by certain data pretreatment methods or even neutral-looking spectral regions can be of great importance for a convolutional neural network. Using a method called Gradient-weighted Class Activation Mapping, we attempt to peer into the black box of convolutional neural networks in microbiological applications and show which Raman spectral regions are responsible for accurate classification.

Live tracking metabolic networks and physiological responses within microbial assemblages at single-cell level.

Microbial interactions impact the functioning of both natural and engineered systems, yet our ability to directly monitor these highly dynamic and spatially resolved interactions in living cells is very limited. Here, we developed a synergistic approach coupling single-cell Raman microspectroscopy with 15N2 and 13CO2 stable isotope probing in a microfluidic culture system (RMCS-SIP) for live tracking of the occurrence, rate, and physiological shift of metabolic interactions in active microbial assemblages. Quantitative and robust Raman biomarkers specific for N2 and CO2 fixation in both model and bloom-forming diazotrophic cyanobacteria were established and cross-validated. By designing a prototype microfluidic chip allowing simultaneous microbial cultivation and single-cell Raman acquisition, we achieved temporal tracking of both intercellular (between heterocyst and vegetative cells of cyanobacteria) and interspecies N and C metabolite exchange (from diazotroph to heterotroph). Moreover, single-cell N and C fixation and bidirectional transfer rate in living cells were quantified via SIP-induced characteristic Raman shifts. Remarkably, RMCS captured physiological responses of metabolically active cells to nutrient stimuli through comprehensive metabolic profiling, providing multimodal information on the evolution of microbial interactions and functions under fluctuating conditions. This noninvasive RMCS-SIP is an advantageous approach for live-cell imaging and represents an important advancement in the single-cell microbiology field. This platform can be extended for real-time tracking of a wide range of microbial interactions with single-cell resolution and advances the understanding and manipulation of microbial interactions for societal benefit.