Unusual dark-field hyperspectral and confocal Raman microscopy features of a nanoporous gold electrode coated with porphyrazine complex.

Nanoporous gold electrodes are of great interest in electroanalytical chemistry, because of their unusual activity and large surface area. The electrochemical activity can be further improved by coating with molecular catalysts such as the tetraruthenated cobalt-tetrapyridylporphyrazines investigated in this work. The plasmonic enhancement of the scattered light at the nanoholes and borders modifies the electrode's optical characteristics, improving the transmission through the surface-enhanced Raman scattering (SERS) effect. When monitored by hyperspectral dark-field and confocal Raman microscopy, this effect allows probing of the porphyrazine species at the plasmonic nanholes, improving the understanding of the chemically modified gold electrodes.

Isotope labeled 3D-Raman confocal imaging and atomic force microscopy study on epithelial cells interacting with the fungus Candida albicans.

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin is responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100-2200 cm-1 attributed to carbon­deuterium bending and at 477 cm-1, attributed to the nitrogen­deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection.

In vivo analysis of the stratum corneum of Japanese neonates and infants using confocal Raman spectroscopy: a pilot study.

BACKGROUND: Physiological skin properties of neonates and infants change drastically after birth and are implicated in the onset of atopic dermatitis and other diseases. Studies have measured physiological skin properties in infants; however, how these properties change over time remains unclear. No reports have measured ceramide in the stratum corneum of infants using confocal Raman spectroscopy; hence, we used it to measure the physiological properties of the skin, including ceramide, in infants. MATERIALS AND METHODS: The water content and other factors in the skin of infants aged 0, 1, and 6 months were measured. All measurements were performed five times indoors at 22 ± 2°C and 50% ± 10% relative humidity in the middle of the calf at 4-µm distances, and their mean was calculated. RESULTS: The water content of the area between the skin surface and superficial layers was the lowest in newborns as compared with other ages, and the deeper the skin layer, the higher the water content. The stratum corneum, evaluated using confocal Raman spectroscopy, was the thickest in newborns and gradually thinned with age. Its water content was the lowest in newborns. The levels of natural moisturizing factor, ceramide, and cholesterol were higher in newborns and tended to decrease with age. CONCLUSION: This report is the first to evaluate ceramide in the stratum corneum of infants using confocal Raman spectroscopy and could help in conducting subsequent longitudinal measurements of physiological skin properties in neonates and infants.

Directional assessment of the skin barrier function in vivo.

INTRODUCTION: The fundamental function of the epidermis is to provide an inside-out barrier to water loss and an outside-in barrier to penetration of external irritants. Transepidermal water loss (TEWL) has been extensively used as a method of estimating the skin barrier quality, typically without any consideration of directionality. The validity of TEWL as an estimate of skin permeability to external substances has been controversial in vitro and in vivo. The aim of this work was to assess the relationship between TEWL and the penetration of a topically applied external marker (caffeine) in healthy skin in vivo before and following a challenge to the barrier. METHODS: The skin barrier was challenged by application of aqueous solutions of mild cleanser products under occlusion for 3 h on the forearms of nine human participants. Skin barrier quality was evaluated before and after the challenge by measuring the TEWL rate and the permeated amount of topically applied caffeine using in vivo confocal Raman microspectroscopy. RESULTS: No skin irritation was observed following the skin barrier challenge. TEWL rates and the caffeine penetrated amount in the stratum corneum after the challenge were not correlated. A weak correlation was observed when the changes were corrected to water-only treatment. TEWL values can be influenced by environmental conditions as well as the skin temperature and water content. CONCLUSIONS: Measuring TEWL rates is not always representative of the outside-in barrier. TEWL may be useful in differentiating large changes in skin barrier function (e.g., between healthy and compromised skin) but is less sensitive to small variations following topical application of mild cleansers.

Transport of hydrocortisone in targeted layers of the skin by multi-lamellar liposomes.

Hydrocortisone (HyC), a hydrophobic pharmaceutical active, was encapsulated in multi-lamellar liposomes (MLLs) composed of P100, a mixture of phospholipids, and Tween®80. Three different HyC-loaded formulations were designed to target the stratum corneum, the living epidermis and the hypodermis. The impact of encapsulation on their size, elasticity and zeta potential, the three key factors controlling MLLs skin penetration, was studied. Raman mapping of phospholipids and HyC allowed the localisation of both components inside an artificial skin, Strat-M®, demonstrating the efficiency of the targeting. Percutaneous permeation profiles through excised human skin were performed over 48 h, supporting results on artificial skin. Their modelling revealed that HyC encapsulated in MLLs, designed to target the stratum corneum and living epidermis, exhibited a non-Fickian diffusion process. In contrast, a Fickian diffusion was found for HyC administered in solution, in a pharmaceutical cream and in transdermal MLLs. These results allowed us to propose a mechanism of interaction between HyC-containing MLLs and the skin.

Multispectral Raman Differentiation of Malignant Skin Neoplasms In Vitro: Search for Specific Biomarkers and Optimal Wavelengths.

Confocal scanning Raman and photoluminescence (PL) microspectroscopy is a structure-sensitive optical method that allows the non-invasive analysis of biomarkers in the skin tissue. We used it to perform in vitro diagnostics of different malignant skin neoplasms at several excitation wavelengths (532, 785 and 1064 nm). Distinct spectral differences were noticed in the Raman spectra of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), compared with healthy skin. Our analysis of Raman/PL spectra at the different excitation wavelengths enabled us to propose two novel wavelength-independent spectral criteria (intensity ratios for 1302 cm-1 and 1445 cm-1 bands, 1745 cm-1 and 1445 cm-1 bands), related to the different vibrational "fingerprints" of cell membrane lipids as biomarkers, which was confirmed by the multivariate curve resolution (MCR) technique. These criteria allowed us to differentiate healthy skin from BCC and SCC with sensitivity and specificity higher than 95%, demonstrating high clinical importance in the differential diagnostics of skin tumors.

Analysis of Milk Microstructure Using Raman Hyperspectral Imaging.

Optical spectroscopic analysis of the chemical composition of milk in its natural state is complicated by a complex colloidal structure, represented by differently sized fat and protein particles. The classical techniques of molecular spectroscopy in the visible, near-, and mid-infrared ranges carry only bulk chemical information about a sample, which usually undergoes a destructive preparation stage. The combination of Raman spectroscopy with confocal microscopy provides a unique opportunity to obtain a vibrational spectrum at any single point of the sample volume. In this study, scanning confocal Raman microscopy was applied for the first time to investigate the chemical microstructure of milk using samples of various compositions. The obtained hyperspectral images of selected planes in milk samples are represented by three-dimensional data arrays. Chemometric data analysis, in particular the method of multivariate curve resolution, has been used to extract the chemical information from complex partially overlaid spectral responses. The results obtained show the spatial distribution of the main chemical components, i.e., fat, protein, and lactose, in the milk samples under study using intuitive graphical maps. The proposed experimental and data analysis method can be used in an advanced chemical analysis of natural milk and products on its basis.

Label-free Raman spectroscopy characterizes signatures of inflammation and fibrosis in the silicosis.

Silicosis is an occupational disease that seriously damages the life and health of miners. Herein, we constructed a mouse model of silicosis and used label-free confocal Raman spectroscopy to analyze the biomolecular variations in lung fibrous nodules and inflammatory sites. The mice were exposed to silica particles for 1 month (SIL-1M group), 3 months (SIL-3M group), or no exposure (control tissues, NS). Raman spectra obtained from treated and untreated lung tissue were subjected to chemometric analysis to quantify biochemical composition differences in the silicosis. Simultaneously, immunohistochemistry and collagen staining were used to evaluate inflammation, fibrosis, and apoptosis. As a result, the SIL-1M and SIL-3M groups showed significant differences in cholesterol, lipids, amino acids, nucleic acids, and cytochrome C, and the collagen peaks at 1248 cm-1 and 1448 cm-1 were significantly higher than in the NS group. Notably, glycogen and phospholipid may be an inflammatory indicator consistent with NF-κB expression. In addition, significant differences in collagen and cytochrome C content in silicosis lung tissue were found using Raman spectroscopy and were verified by Masson's staining and Bax/Bcl-2 expression ratio. In summary, our findings provide a label-free technique to understand the biochemical changes in lung inflammatory and fibrosis microenvironment after exposure to silica particles and provide a valuable reference for studying the mechanism of silicosis.

Trans-Resveratrol Decreases Membrane Water Permeability: A Study of Cholesterol-Dependent Interactions.

Resveratrol (RSV), a biologically active plant phenol, has been extensively investigated for cancer prevention and treatment due to its ability to regulate intracellular targets and signaling pathways which affect cell growth and metastasis. The non-specific interactions between RSV and cell membranes can modulate physical properties of membranes, which in turn can affect the conformation of proteins and perturb membrane-hosted biological functions. This study examines non-specific interactions of RSV with model membranes having varying concentrations of cholesterol (Chol), mimicking normal and cancerous cells. The perturbation of the model membrane by RSV is sensed by changes in water permeability parameters, using Droplet Interface Bilayer (DIB) models, thermotropic properties from Differential Scanning Calorimetry, and structural properties from confocal Raman spectroscopy, all of which are techniques not complicated by the use of probes which may themselves perturb the membrane. The nature and extent of interactions greatly depend on the presence and absence of Chol as well as the concentration of RSV. Our results indicate that the presence of RSV decreases water permeability of lipid membranes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), indicating a capability for RSV in stiffening fluidic membranes. When Chol is present, however, (at 4:1 and 2:1 mol ratio DOPC to cholesterol), the addition of RSV has no significant effect upon the water permeability. DSC thermograms show that RSV interacts with DOPC and DOPC/Chol bilayers and influences their thermotropic phase behavior in a concentration-dependent manner, by decreasing the main phase transition temperature and enthalpy, with a phase separation shown at the higher concentrations of RSV. Raman spectroscopic studies indicate an ordering effect of RSV on DOPC supported bilayer, with a lesser extent of ordering in the presence of Chol. Combined results from these investigations highlight a differential effect of RSV on Chol-free and Chol-enriched membranes, respectively, which results constitute a bellwether for increased understanding and effective use of resveratrol in disease therapy including cancer.

ex vivo-in vivo comparison of drug penetration analysis by confocal Raman microspectroscopy and tape stripping.

When it comes to skin penetration analysis of a topically applied formulation, the number of suitable methods is limited, and they often lack in spatial resolution. In vivo studies are pivotal, especially in the approval of a new product, but high costs and ethical difficulties are limiting factors. For that reason, good ex vivo models for testing skin penetration are crucial. In this study, caffeine was used as a hydrophilic model drug, applied as a 2% (w/w) hydrogel, to compare different techniques for skin penetration analysis. Confocal Raman microspectroscopy (CRM) and tape stripping with subsequent HPLC analysis were used to quantify caffeine. Experiments were performed ex vivo and in vivo. Furthermore, the effect of 5% (w/w) 1,2-pentanediol on caffeine skin penetration was tested, to compare those methods regarding their effectiveness in detecting differences between both formulations.

Application of Confocal Raman Microscopy for the Characterization of Topical Semisolid Formulations and their Penetration into Human Skin Ex Vivo.

PURPOSE: The quality testing and approval procedure for most pharmaceutical products is a streamlined process with standardized procedures for the determination of critical quality attributes. However, the evaluation of semisolid dosage forms for topical drug delivery remains a challenging task. The work presented here highlights confocal Raman microscopy (CRM) as a valuable tool for the characterization of such products. METHODS: CRM, a laser-based method, combining chemically-selective analysis and high resolution imaging, is used for the evaluation of different commercially available topical acyclovir creams. RESULTS: We show that CRM enables the spatially resolved analysis of microstructural features of semisolid products and provides insights into drug distribution and polymorphic state as well as the composition and arrangement of excipients. Further, we explore how CRM can be used to monitor phase separation and to study skin penetration and the interaction with fresh and cryopreserved excised human skin tissue. CONCLUSION: This study presents a comprehensive overview and illustration of how CRM can facilitate several types of key analyses of semisolid topical formulations and of their interaction with their biological target site, illustrating that CRM is a useful tool for research, development as well as for quality testing in the pharmaceutical industry.

Spatiotemporal distribution of chemical signatures exhibited by Myxococcus xanthus in response to metabolic conditions.

Myxococcus xanthus is a common soil bacterium with a complex life cycle, which is known for production of secondary metabolites. However, little is known about the effects of nutrient availability on M. xanthus metabolite production. In this study, we utilize confocal Raman microscopy (CRM) to examine the spatiotemporal distribution of chemical signatures secreted by M. xanthus and their response to varied nutrient availability. Ten distinct spectral features are observed by CRM from M. xanthus grown on nutrient-rich medium. However, when M. xanthus is constrained to grow under nutrient-limited conditions, by starving it of casitone, it develops fruiting bodies, and the accompanying Raman microspectra are dramatically altered. The reduced metabolic state engendered by the absence of casitone in the medium is associated with reduced, or completely eliminated, features at 1140 cm-1, 1560 cm-1, and 1648 cm-1. In their place, a feature at 1537 cm-1 is observed, this feature being tentatively assigned to a transitional phase important for cellular adaptation to varying environmental conditions. In addition, correlating principal component analysis heat maps with optical images illustrates how fruiting bodies in the center co-exist with motile cells at the colony edge. While the metabolites responsible for these Raman features are not completely identified, three M. xanthus peaks at 1004, 1151, and 1510 cm-1 are consistent with the production of lycopene. Thus, a combination of CRM imaging and PCA enables the spatial mapping of spectral signatures of secreted factors from M. xanthus and their correlation with metabolic conditions.

Facile Fabrication of Anisotropic Multicompartmental Microfibers Using Charge Reversal Electrohydrodynamic Co-Jetting.

Anisotropic microstructures are utilized in various fields owing to their unique properties, such as reversible shape transitions or on-demand and sequential release of drug combinations. In this study, anisotropic multicompartmental microfibers composed of different polymers are prepared via charge reversal electrohydrodynamic (EHD) co-jetting. The combination of various polymers, such as thermoplastic polyurethane, poly(D,L-lactide-co-glycolide), poly(vinyl cinnamate), and poly(methyl methacrylate), results in microfibers with distinct compositional boundaries. Charge reversal during EHD co-jetting enables facile fabrication of multicompartmental microfibers with the desired composition and tunable inner architecture, broadening their spectrum of potential applications, such as functional microfibers and cell scaffolds with multiple physical and chemical properties.

Nutrient limitation regulates the properties of extracellular electron transfer and hydraulic shear resistance of electroactive biofilm.

Understanding the roles of nutrient restriction in extracellular electron transfer (EET) and stability of mixed electroactive biofilm is essential in pollutant degradation and bioenergy production. However, the relevant studies are still limited so far. Herein, the effect of nutrient restriction on the EET pathways and stability of mixed electroactive biofilm was explored. It was found that the electroactive Pseudomonas and Geobacter genera were selectively enriched in the biofilms cultured under total nutrient and P-constrained conditions, and two EET pathways including direct and indirect were found, while Rhodopseudomonas genus was enriched in the N-constrained biofilm, which only had the direct EET pathway. Moreover, multiple analyses including 2D confocal Raman spectra revealed that P-constrained biofilm was rich in extracellular polymeric substances (EPS) especially for polysaccharide, presented a dense and uniform layered distribution, and had better stability than N-constrained biofilm with lower EPS and biofilm with heterostructures cultured under total nutrient conditions.

To study the effect of acute infrared radiation-induced alterations in human skin at cellular and molecular level using in vivo confocal Raman spectroscopy.

BACKGROUND: Solar radiations are classified in terms of wavelengths, including visible light, infrared, and ultraviolet. Infrared radiation (IR) accounts the largest proportion of solar radiations that cause oxidative stress-induced aging of human skin. This study investigates the biochemical changes in proteins, lipids, and DNA associated with acute exposure to IR radiations. METHOD: In vivo confocal Raman spectroscopy was used to examine the forearms region of 20 healthy participants with phototype II skin, aged between 18 and 30 years, without IR incidence (T0), with IR incidence 30 minutes (T30) at day 1 and 30 minutes at day 2 (T60). One-way ANOVA and two-tailed t test along with post hoc Bonferroni correction were used to detect the existence of significant differences in the timestamps of stratum corneum, stratum basale, and dermis at all IR wavenumbers under test. RESULTS: An increase in the Raman peaks of stratum corneum lipids, decrease in stratum basal DNA peaks, and a shift in the amide I peak of collagen in the skin dermis were observed. One-way ANOVA results showed significant differences among timestamps of stratum corneum, stratum basale, and dermis at all wavenumbers under test (P < .001). Furthermore, paired timestamps also showed significant differences (P < .016) except at two wavenumbers 1293 cm-1 and 852 cm-1 in stratum corneum and basale layer clusters on timestamps (T0 & T30 and T30 & T60, P > .016). This study proved that confocal Raman spectroscopy is an useful technique for early evaluation of IR-induced skin changes.

Confocal Raman spectroscopy is suitable to assess hair cleansing-derived skin dryness on human scalp.

BACKGROUND: The purpose of this pilot study was to provide information about the washout-dependent depletion of important skin components in the horny layer of the scalp. They were taken as markers for scalp drying effects of cosmetic cleansing products and were measured directly in vivo. METHOD: In vivo confocal Raman spectroscopy was used to measure the depletion of the total natural moisturizing factor (total NMF) and some of its components (urea and lactic acid) as well as a fraction of stratum corneum lipids, after repeated washing with a standard shampoo on the human scalp. RESULTS: The measurements showed a reduction in the amount of NMF and lipids of the stratum corneum caused by repeated shampooing. CONCLUSION: Confocal Raman spectroscopy is an innovative technology that can be used successfully in vivo on the hairy scalp. The loss of the most important skin components caused by hair washing can be quantified directly with this technology. The method is valuable to support the development cosmetic cleansing products, as it is suitable to directly compare the effects of different product candidates on the human scalp in a most realistic way.

Characterization and ex vivo evaluation of excised skin samples as substitutes for human dermal barrier in pharmaceutical and dermatological studies.

BACKGROUND: Excised animal and human skins are frequently used in permeability testing in pharmaceutical research. Several factors exist that may have influence on the results. In the current study some of the skin parameters that may affect drug permeability were analysed for human, mouse, rat and pig skin. MATERIALS AND METHODS: Classic biophysical skin parameters were measured (e.g. pH, hydration, permittivity, transepidermal water loss). Physiological characteristics of the skins were also analysed by confocal Raman spectroscopy, scanning electron microscopy and two-photon microscopy. RESULTS: Based on biophysical testing, skin barrier function was damaged in psoriatic mouse skin and in marketed pig skin. Hydration and pH values were similar among the species, but freezing and thawing reduced the water content of the skins and shifted the surface pH to acidic. Aging reduced hydration and permittivity, resulting in impaired barrier function. Mechanical sensitization used in permeability studies resulted in proportional thinning of dead epidermis. DISCUSSION: Results indicate that depending on the scientific question it should be considered whether fresh or frozen tissue is used, and for certain purposes rodent skins are well usable. The structure of the skin tissue (ceramide, cholesterol, keratin, natural moisturizing factor or urea) is similar in rats and mice, but due to the higher skin thickness the lipid distribution is different in porcine skin. Psoriasis led to irregular chemical composition of the skin. CONCLUSION: A comprehensive evaluation of skin samples of four species was performed. The biophysical and microscopic observations should be considered when selecting drug penetration models and experimental conditions.

Versatile Confocal Raman Imaging Microscope Built from Off-the-Shelf Opto-Mechanical Components.

Confocal Raman microscopic (CRM) imaging has evolved to become a key tool for spatially resolved, compositional analysis and imaging, down to the µm-scale, and nowadays one may choose between numerous commercial instruments. That notwithstanding, situations may arise which exclude the use of a commercial instrument, e.g., if the analysis involves toxic or radioactive samples/environments; one may not wish to render an expensive instrument unusable for other uses, due to contamination. Therefore, custom-designed CRM instrumentation-being adaptable to hazardous conditions and providing operational flexibility-may be beneficial. Here, we describe a CRM setup, which is constructed nearly in its entirety from off-the-shelf optomechanical and optical components. The original aim was to develop a CRM suitable for the investigation of samples exposed to tritium. For increased flexibility, the CRM system incorporates optical fiber coupling to both the Raman excitation laser and the spectrometer. Lateral raster scans and axial profiling of samples are facilitated by the use of a motorized xyz-translation assembly. Besides the description of the construction and alignment of the CRM system, we also provide (i) the experimental evaluation of system performance (such as, e.g., spatial resolution) and (ii) examples of Raman raster maps and axial profiles of selected thin-film samples (such as, e.g., graphene sheets).

Machine Learning Assisted Handheld Confocal Raman Micro-Spectroscopy for Identification of Clinically Relevant Atopic Eczema Biomarkers.

Atopic dermatitis (AD) is a common chronic inflammatory skin dermatosis condition due to skin barrier dysfunction that causes itchy, red, swollen, and cracked skin. Currently, AD severity clinical scores are subjected to intra- and inter-observer differences. There is a need for an objective scoring method that is sensitive to skin barrier differences. The aim of this study was to evaluate the relevant skin chemical biomarkers in AD patients. We used confocal Raman micro-spectroscopy and advanced machine learning methods as means to classify eczema patients and healthy controls with sufficient sensitivity and specificity. Raman spectra at different skin depths were acquired from subjects' lower volar forearm location using an in-house developed handheld confocal Raman micro-spectroscopy system. The Raman spectra corresponding to the skin surface from all the subjects were further analyzed through partial least squares discriminant analysis, a binary classification model allowing the classification between eczema and healthy subjects with a sensitivity and specificity of 0.94 and 0.85, respectively, using stratified K-fold (K = 10) cross-validation. The variable importance in the projection score from the partial least squares discriminant analysis classification model further elucidated the role of important stratum corneum proteins and lipids in distinguishing two subject groups.

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum.

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR-partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400-1500 cm-1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.