Use Of Confocal Raman Microscopy Research Articles

The use of confocal Raman microscopy and microfluidic channels to monitor the location and mobility of β-carotene incorporated in droplet-stabilized oil-in-water emulsions

This study sought to explore the combined use of confocal Raman microscopy and microfluidic channels to probe the location and mobility of hydrophobic antioxidant (β-carotene) incorporated at the interface of food-grade droplet-stabilized emulsions (DSEs).Microfluidic channels were used to isolate emulsion droplets for efficient investigation of antioxidant mobility. This approach proved more conclusive than fixing the sample in agarose, because a single layer of droplets could be obtained. Results also indicated that the migration of β-carotene incorporated in shell droplets of olive oil and trimyristin DSEs to core droplets was minimal and beta-carotene remained mostly localised at the interface even after 3 days of production.This work demonstrates that microfluidic isolation of emulsion droplets combined with confocal Raman microscopy can give new insights into the spatial variation of chemical composition within emulsions.This study revealed that the migration of β-carotene between shell and core was minimal and hence it may be possible to concurrently deliver two incompatible compounds by spatially segregating them between shell and core compartments of DSEs.

Analysis of Transdermal Patch Layers via Confocal Raman Microscopy

A transdermal patch is a method of drug delivery through the skin into the bloodstream that is composed of layers that store and release the active pharmaceutical ingredient (API) over time. The ability to analyze these layers without damaging the patch allows for quality control and stability measurements. Raman spectroscopy has excellent chemical identification abilities, thanks to the fingerprint like spectrum it produces, that deliver rapid discrimination between components typically found in transdermal patches, such as polymers and APIs. Coupling this with a confocal microscope gives high spatial resolution on all three axes, with the pinhole providing otherwise unachievable axial resolution, permitting depth profile analysis. Using this technique, the patch can be analyzed from top to bottom without any risk of damage. This article highlights the use of confocal Raman microscopy to acquire a 3D Raman map of a transdermal patch for layer evaluation. The map revealed six layers which were composed of four polymers and the API layer at the center of the patch.

A multitechnique approach for discrimination and identification of lipsticks for forensic purposes.

Cosmetics are becoming more and more popular; consequently, the chance of finding them as microtraces at a crime scene increases. They are easily transferable and can provide a link between a suspect and a victim. For this reason, identifying and comparative analysis of red lipstick - the most popular and used - is required. The aim of this study was to apply a multitechnique methodology for the comparative forensic analysis of the red lipsticks traces of a very similar hue. For this purpose, four methods of different physicochemical basics - two nondestructive spectroscopic and two destructive separation techniques - were used. The possibilities and advantages of attenuated total reflection infrared spectroscopy (ATR-FTIR), confocal Raman microscopy (CRM), capillary electrophoresis (MEKC) and gas chromatography-mass spectrometry (GC-MS) have been combined. Specially prepared lipstick traces in various forms (imprints, smears) on different surfaces (absorbent and nonabsorbent) were analyzed to confirm the usefulness of the proposed methods. The premise is that if two methods yield a consistent result, the investigation is terminated at this stage. All investigated traces were properly identified. First, the ATR-FTIR method as a nondestructive technique is recommended. Sometimes, due to strong interferences from the substrates, the newly proposed method with the use of confocal Raman microscopy may be an alternative. The next recommendation is the MEKC method. Only in case of the absence of unambiguous conclusions, it is proposed to use the GC-MS method. This methodology has the potential to be applied in the comparative analysis of red lipsticks for forensic purposes.

Vibrational Spectroscopy in the Study of Composite and Nanostructured Materials for Electrochemistry

Our research group has recently demonstrated the ability to probe within composite membranes formed by sandwiching single layer graphene (SLG) between two Nafion layers (Nafion 211, 25 µm thickness). These graphene-sandwich membranes have shown high selectivity for proton transport across electrochemical cells [1,2]. Using a microscope equipped with an oil-immersion objective, the graphene layer is readily detected in confocal Raman microscopy depth-profiles (z-direction profiles) of the composite membranes. Spectra are sensitive to defects and stress within the buried graphene layer. This presentation will discuss the use of confocal Raman microscopy for structural characterization and spatial profiling in the development of composite polymer electrolyte membranes that contain a graphene layer as a barrier to ion transport. In a second application area, infrared spectroscopy is being adapted to investigate properties of Nafion ionomer particles in dilute dispersions and during transformation to gel and solid phases through the use of time-resolved attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Since the properties of dispersing fluids can affect the mechanical strength of solution-cast Nafion thin films [3], it is of interest to gain insights into ionomer properties in the dispersion and during phase changes. A well-resolved peak associated with vibrational motions of the Nafion side chain -SO3 -end group is shown to be sensitive to gel-phase development. The persistence of hydration water in the vicinity of the -SO3 -group helps maintain a constant local refractive index that limits optical distortion in the band as the ionomer assembles into the phase-separated framework structure of the solid membrane. ATR FTIR measurements predict trends in evaporative water loss from the dispersion and entry into the gel-phase consistent with expectations of gravimetric measurements and earlier studies [3] of Nafion ionomer dispersion properties. 1. Bukola, S., et al. J. Am. Chem. Soc. 2018, 140, 1743.2. Bukola, S., et al., ACS Appl. Nano Mater. 2019, 2, 964.3. Kim, Y.S., et al., Macromolecules 2015, 48, 2161.

Use of confocal Raman microscopy to characterise ethyl cyanoacrylate adhesive depth curing.

In situ spatial temporal measurement of monomer conversion during adhesive bondline curing remains a challenging area. The aim of this work was to demonstrate the effectiveness of using confocal Raman microscopy in a specially configured experimental set-up, as a versatile tool for measuring monomer concentration changes as a function of both time and adhesive bond depth during ethyl cyanoacrylate polymerisation. This also allowed monitoring of the extent of polymerisation at the adhesive substrate interface independently of the bulk bondline polymerisation region. Key kinetic parameters such as inhibition time tlag, rate of reaction Rmax and extent of reaction [αt]max were obtained by fitting the experimental data to sigmoidal growth curves using simple piecewise regression models. A systematic characterisation of a polymerisation reaction was conducted using different sample substrate types (copper alloy (red brass), aluminium, aluminium alloy, stainless steel and borosilicate glass) and at various reaction temperatures. Reaction rates were found to decrease further away from the substrate interface in the bulk volume region. The fastest kinetics occurred in the vicinity of nucleophilic hydroxyl rich surfaces such as at the copper alloy (red brass). In addition to substrate surface chemistry, surface roughness was also a factor, with the highest reaction rates occurring with a grit blasted (roughened) aluminium alloy (2024 T3) surface. An approximately linear dependence of the ln Rmaxvs. 1/T (Arrhenius) plot was recorded within the temperature range of 291-328 K. A better fit was obtained however through the use of two separate linear slopes, possibly indicative of a change of polymerisation reaction mechanism taking place at elevated temperatures with two distinct activation energies. Further work conducted using a larger number of temperatures would be useful to verify this finding. This work confirmed that differences in the rates of interfacial and bulk polymerisation processes could be readily measured in situ using confocal Raman microscopy which is a powerful technique for investigating such surface-confined and bulk polymerisation reactions.

Local hydration in ionomer composite membranes determined with confocal Raman microscopy

Water management in electrochemical energy applications like fuel cells has a crucial impact on performance, in particular on the ionic conduction of ionomer membranes. To strengthen the understanding of water management in such devices, we report a novel method for non-destructive measurements of the hydration of composite membranes based on confocal Raman microscopy. Composite membranes were produced by spray-coating of Nafion into a mesh of electrospun poly(vinylidene fluoride-co-hexafluoropropylene)/polyvinylpyrrolidone (PVDF-HFP/PVP) blend nanofibers. Hydration levels of several pure nanofiber meshes and nanofiber/Nafion composites were evaluated by linear least squares fitting of reference Raman spectra to hyperspectral images. We found that spectral contribution of water to nanofiber spectra depends on the PVDF-HFP/PVP ratio and is independent from fiber diameter. Further, we were able to reliably determine nanofiber polymer composition of single fibers based on Raman spectroscopy. Raman imaging of composite membranes was performed at ambient air and fully hydrated conditions to study the local hydration in PVDF-HFP/PVP/Nafion composites as well as in a Nafion XL membrane. 2D through-plane mappings revealed that the nanofiber hydration positively correlated with PVP content. In the Nafion XL membrane, the polytetrafluoroethylene-based reinforcement was verified as a hydrophobic layer sandwiched between Nafion ionomer, which showed a more than 10% reduced hydration compared to the outer Nafion layers. These results motivate the use of confocal Raman microscopy as a novel method to investigate the local water distribution in ionomer composite membranes that are widely used in electrochemical energy conversion.

Confocal Raman Microscopy for the Determination of Protein and Quaternary Ammonium Ion Loadings in Biocatalytic Membranes for Electrochemical Energy Conversion and Storage.

The need to immobilize active enzyme, while ensuring high rates of substrate turnover and electronic charge transfer with an electrode, is a centrally important challenge in the field of bioelectrocatalysis. In this work, we demonstrate the use of confocal Raman microscopy as a tool for quantitation and molecular-scale structural characterization of ionomers and proteins within biocatalytic membranes to aid in the development of energy efficient biofuel cells. A set of recently available short side chain Aquivion ionomers spanning a range of equivalent weight (EW) suitable for enzyme immobilization was investigated. Aquivion ionomers (790 EW, 830 EW and 980 EW) received in the proton-exchanged (SO3H) form were treated with tetra-n-butylammonium bromide (TBAB) to neutralize the ionomer and expand the size of ionic domains for enzyme incorporation. Through the use of confocal Raman microscopy, membrane TBA+ ion content was predicted in calibration studies to within a few percent of the conventional titrimetric method across the full range of TBA+: SO3- ratios of practical interest (0.1 to 1.7). Protein incorporation into membranes was quantified at the levels expected in biofuel cell electrodes. Furthermore, features associated with the catalytically active, enzyme-coordinated copper center were evident between 400 and 500 cm-1 in spectra of laccase catalytic membranes, demonstrating the potential to interrogate mechanistic chemistry at the enzyme active site of biocathodes under fuel cell reaction conditions. When benchmarked against the 1100 EW Nafion ionomer in glucose/air enzymatic fuel cells (EFCs), EFCs with laccase air-breathing cathodes prepared from TBA+ modified Aquivion ionomers were able to reach maximum power densities (Pmax) up to 1.5 times higher than EFCs constructed with cathodes prepared from TBA+ modified Nafion. The improved performance of EFCs containing the short side chain Aquivion ionomers relative to Nafion is traced to effects of ionomer ion-exchange capacity (IEC, where IEC = EW-1), where the greater density of SO3- moieties in the Aquivion materials produces an environment more favorable to mass transport and higher TBA+ concentrations.

Confocal Raman Microscopy Tracks Cutaneous Delivery of Flufenamic Acid using Lipophilic Versus Hydrophilic Penetration Enhancers

Recent technological advances have led to an increase in the use of confocal Raman microscopy to tackle biophysical and pharmacological issues in topical delivery and dermatology. Advantages of the technique include the ability to optically section intact, full thickness skin without the need to microtome or otherwise physically segment the skin. In addition, both exogenous agents and perturbations to endogenous structure can be monitored without the introduction of probe molecules. In the current work, flufenamic acid (FluA), a non-steroidal anti-inflammatory drug, is a model agent for investigating the influence of lipophilic versus hydrophilic penetration enhancers on the outer barrier layer of skin. For topical delivery to be effective, a drug must cross the stratum corneum (SC) barrier into viable tissue. In separate experiments, FluA in octanol or propylene glycol/ethanol (75/25) is applied to skin for varying times followed by confocal Raman microscopic mapping of drug and enhancer spatial distribution. The FluA pathway is tracked by the C=C stretching mode at 1618 cm−1. The exogenous enhancers are spectroscopically differentiated from the endogenous lipids by utilizing deuterated versions of the enhancers which do not affect penetration parameters. Discrete pockets (5-20 μm) of both enhancers are observed throughout the SC. High concentrations of FluA are co-localized with octanol inclusions which appear to provide a pathway to the viable epidermis for the drug. In contrast, FluA concentrates in the upper SC when using the hydrophilic agent and endogenous lipids are unperturbed in regions outside the enhancer pockets. The ability to examine perturbations to endogenous ultrastructure and molecular structure in skin while tracking penetration pathways provides insight into delivery mechanisms.

Raman imaging processed cheese and its components

Controlling the quality of cheese involves monitoring its taste, texture, stability and appearance. One method of doing so involves the analysis of cheese microstructure. This research investigated the use of confocal Raman microscopy for observing the microstructure of processed cheese including the distribution of additives therein. Reference Raman spectra were obtained for several additives commonly present in processed cheese including: trisodium citrate (TSC), sorbic acid, paprika and corn starch. Low and high spatial resolution Raman images were obtained from commercial and custom formulated processed cheeses. These were created using band integrals and principal component analysis scores values. Results of this investigation show that fat, protein, water, TSC, paprika and starch distributions can be imaged effectively using the many Raman imaging methods, including high spatial resolution Raman microscopy. Copyright © 2016 John Wiley & Sons, Ltd.

The microstructure and component distribution in spray-dried emulsion particles

Microencapsulation by spray drying of oil-in-water (o/w) emulsions provides a means to encapsulate functional lipophilic ingredients. The active ingredient is dispersed in continuous solid phase providing protection. However, the encapsulation efficiency depends on the microstructure and morphology of the dry particles influenced by several mechanisms occurring during processing such as oil droplet breakup during atomization, ingredient diffusivity, interfacial adsorption of surface active ingredients, and drying kinetics. In this work, sunflower oil (model for lipophilic compounds) was encapsulated in solid particles composed of acacia gum and maltodextrin DE12. Three powders with different initial emulsion size (e.g. about 0.1 and 1 μm) and atomized under high and low shear rate were analysed for the morphology and distribution of oil droplets and matrix constituents within the solid particle (20–100 μm). The microscopic (optical, SEM, LVSEM, confocal Raman), spectroscopic (XPS) and analytical (solvent extraction) techniques used were either qualitative or quantitative. Their combination made it possible to determine both the composition at the surface and inside the particle. The surface differs from the bulk in composition, confirming the constituent segregation during spray drying, and depended on the initial emulsion size and atomization conditions that must be controlled for an efficient encapsulation. Especially, the use of confocal Raman microscopy is promising for the study of processstructure-properties relationship.

Comment on “Observation of mutual diffusion of macromolecules in PS/PMMA binary films by confocal Raman microscopy” by C. Hu, X. Chen, J. Chen, W. Zhang and M. Q. Zhang, Soft Matter, 2012,8, 4780

A paper by Hu et al. (Soft Matter, 2012, 8, 4780) reports on the use of confocal Raman microscopy to resolve mutual diffusion between polystyrene (PS) and poly(methyl methacrylate) (PMMA). In-depth optical sectioning is employed to measure the diffusive broadening of the originally planar PS-PMMA interface, from which tracer and mutual diffusion coefficients and values for the PS-PMMA thermodynamic interaction parameter are extracted. Here, a reinterpretation of Hu's data that leads to a completely different scenario is presented, as apparent diffusive broadening can be mostly attributed to optical distortions inherent to the probe methodology. It also explains the lack of consistency of kinetic and thermodynamic parameters obtained by the authors from their diffusion analysis in comparison with earlier published data on this system. Overall, it highlights the importance of carrying out appropriate data analysis when confocal Raman microscopy is applied in dry depth-profiling investigations.

Quantification of whey in fluid milk using confocal Raman microscopy and artificial neural network

In this work, we assessed the use of confocal Raman microscopy and artificial neural network as a practical method to assess and quantify adulteration of fluid milk by addition of whey. Milk samples with added whey (from 0 to 100%) were prepared, simulating different levels of fraudulent adulteration. All analyses were carried out by direct inspection at the light microscope after depositing drops from each sample on a microscope slide and drying them at room temperature. No pre- or posttreatment (e.g., sample preparation or spectral correction) was required in the analyses. Quantitative determination of adulteration was performed through a feed-forward artificial neural network (ANN). Different ANN configurations were evaluated based on their coefficient of determination (R2) and root mean square error values, which were criteria for selecting the best predictor model. In the selected model, we observed that data from both training and validation subsets presented R2>99.99%, indicating that the combination of confocal Raman microscopy and ANN is a rapid, simple, and efficient method to quantify milk adulteration by whey. Because sample preparation and postprocessing of spectra were not required, the method has potential applications in health surveillance and food quality monitoring.

Confocal Raman spectroscopy to trace lipstick with their smudges on different surfaces.

Lipsticks are very popular cosmetic products that can be transferred by contact to different surfaces, being important forensic evidence with an intricate analysis if they are found in a crime scene. This study evaluates the use of confocal Raman microscopy at 780 nm excitation wavelength for the nondestructive identification of 49 lipsticks of different brands and colors, overcoming the lipstick fluorescence problem reported by previous works using other laser wavelengths. Although the lipsticks samples showed some fluorescence, this effect was not so intense to completely overwhelm the Raman spectra. Lipsticks smudges on twelve different surfaces commonly stained with these samples were also analyzed. In the case of the surfaces, some of them provided several bands to the smudge spectra compromising the identification of the lipstick. For these samples spectral subtraction of the interfering bands from the surface was performed. Finally, five different red lipsticks with very similar color were measured on different surfaces to evaluate the lipstick traceability with their smudges even on interfering surfaces. Although previous spectral subtraction was needed in some cases, all the smudged were linked to their corresponding lipsticks even when they are smeared on the interfering surfaces. As a consequence, confocal Raman microscopy using the 780 nm excitation laser is presented as a nondestructive powerful tool for the identification of these tricky samples.

Laser‐induced volume changes during confocal Raman microscopy of whey‐protein‐stabilized emulsions and their relationship to protein content and particle diameters

The use of confocal Raman microscopy is applied to the study of whey‐protein‐stabilized oil‐in‐water emulsions. The stability of emulsions is an important parameter for many applications and is dependent on several factors including protein content and particle size. Emulsions were prepared with varying particle size ranging from 0.8 to 10.0 µm. Changes in sample volume due to laser‐induced heating were monitored and correlated to protein content in samples and to particle diameters. It was found that samples with the lowest protein content and larger particle diameters experienced the largest volume changes. The newly developed approach is of a potential predictive use. Copyright © 2013 John Wiley & Sons, Ltd.

Confocal Raman Microscopy of Protein Adsorbed in Chromatographic Particles

Confocal Raman microscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure conformation and distribution of protein adsorbed in wetted chromatographic particles. Monoclonal antibody was loaded into the Fractogel EMD SO(3) (M) cation exchanger at 2 mS/cm or 10 mS/cm. Amide I and III frequencies in the Raman spectrum of the adsorbed protein suggest that there are no detectable changes of the original β-sheet conformation in the chromatographic particles. Protein depth profile measurements indicate that, when the conductivity is increased from 2 mS/cm to 10 mS/cm, there is a change in mass transport mechanism for protein adsorption, from the shrinking-core model to the homogeneous-diffusion model. In this study, the use of confocal Raman microscopy to measure protein distribution in chromatographic particles fundamentally agrees with previous confocal laser scanning microscopic investigations, but confocal Raman spectroscopy enjoys additional advantages: use of unlabeled protein to eliminate fluorescent labeling, ability for characterization of protein secondary structure, and ability for spectral normalization to provide a nondestructive experimental approach to correct light attenuation effects caused by refractive index (RI) mismatching in semiopaque chromatographic particles.

Cellular Uptake and Nanoscale Localization of Gold Nanoparticles in Cancer Using Label-Free Confocal Raman Microscopy

This work demonstrates the use of confocal Raman microscopy (CRM) to measure the dynamics of cellular uptake and localization of gold nanoparticles (GNP) with nanoscale resolution. This is important as nanoparticle cellular interactions are increasingly under investigation to support applications as diverse as drug delivery, gene transfection and a variety of heat and radiation based therapeutics. At the heart of these applications is a need to know the dynamics of nanoparticle cellular uptake and localization (i.e., cell membrane, cytoplasm or nucleus). This process can change dramatically based on size, charge, shape and ligand attached to the nanoparticle. While electron microscopy, atomic emission spectroscopy and histology can be used to assess cellular uptake, they are labor intensive and post-mortem and can miss critical dynamics of the process. For this reason investigators are increasingly turning to optically active nanoparticles that allow direct microscopic interrogation of uptake. Here we show that CRM adds to this evolving armamentarium as a fast, noninvasive, and label-free technique to dynamically study cellular uptake of GNPs with subcellular detail in cancer. Raman laser interaction with GNPs inside cells shows unique spectroscopic features corresponding to the intracellular localization of GNPs over 2 to 24 h at the membrane, cytoplasm or nucleus that are separately verified by histology (silver staining) and electron microscopy. These results show that CRM has the potential to facilitate high-throughput study of the dynamics and localization of a variety of GNPs in multiple cell types.

Confocal Raman Microscopy as a Tool to Investigate Concentration Profiles of Melt Crystallized Ibuprofen/Carnauba Wax

AbstractCoatings are of great significance for pharmaceutical solid dosage forms. They fulfil a number of functions and are often necessary to control drug delivery, to mask bitter taste, or to protect the active pharmaceutical ingredient from detrimental environmental factors. The process of self‐coating by melt crystallization of a suitable binary mixture eliminates the need for an additional process step in the manufacture of a solid drug. Self‐coating relies upon the physical and spatial separation of individual components in a melt during solidification. This paper focuses on the use of confocal Raman microscopy as a nondestructive technique for quantifying the spatial distribution of the components in self‐coated pastilles manufactured from the binary system ibuprofen/carnauba wax. Pastilles are produced from the melt. Raman spectroscopy allows the direct analysis of concentration profiles across the surface of the pastille. Here, the samples are cleaved and the cleaved surface is investigated in order to establish the distribution of the components in the interior of the solid. A univariate calibration model was developed and statistically validated with standard mixtures of ibuprofen and carnauba wax. Different regression models (linear or polynomial, using different significant peaks for the respective compounds) were assessed and a linear model was found to be adequate to determine the concentration gradient in the pastilles.

Optical trapping of unilamellar phospholipid vesicles: investigation of the effect of optical forces on the lipid membrane shape by confocal-Raman microscopy.

Optical trapping of liposomes is a useful tool for manipulating these lipid vesicles for sampling, mechanical testing, spectroscopic observation, and chemical analysis. Through the use of confocal Raman microscopy, this study addresses the effects of optical forces on the structure of unilamellar, dipalmitoylphosphatidylcholine (DPPC) vesicles, both optically trapped in solution and adhered to a coverslip. The energy and forces involved in optical trapping of lipid vesicles were derived in terms of the dielectric contrast between the phospholipid membrane and the surrounding solution; reflection forces at the membrane/water interface were found to be negligible. At optical powers of 9 mW and greater, unilamellar liposomes trapped in bulk solution experience a gradient force sufficiently strong to bend the vesicle membrane, so that a second bilayer from the same vesicle is drawn into the optical trap, with an energy of approximately 6 x 10(-13) erg. For vesicles adhered to a coverslip, the confocal probe can be scanned through the attached vesicle. Optical forces are insufficient to detach the bilayer that is adhered to the glass; however, the upper DPPC bilayer can be manipulated by the optical trap and the shape of the vesicle distorted from a spherical geometry. The effect of calcium ion on the flexibility of membrane bilayers was also tested; with 5 mM calcium ion in solution, the lipid bilayer of a surface-attached liposome is sufficiently rigid so that it cannot be distorted at moderate laser powers.

Electrographic recording

Lipsticks are very popular cosmetic products that can be transferred by contact to different surfaces, being important forensic evidence with an intricate analysis if they are found in a crime scene. This study evaluates the use of confocal Raman microscopy at 780 nm excitation wavelength for the nondestructive identification of 49 lipsticks of different brands and colors, overcoming the lipstick fluorescence problem reported by previous works using other laser wavelengths. Although the lipsticks samples showed some fluorescence, this effect was not so intense to completely overwhelm the Raman spectra. Lipsticks smudges on twelve different surfaces commonly stained with these samples were also analyzed. In the case of the surfaces, some of them provided several bands to the smudge spectra compromising the identification of the lipstick. For these samples spectral subtraction of the interfering bands from the surface was performed. Finally, five different red lipsticks with very similar color were measured on different surfaces to evaluate the lipstick traceability with their smudges even on interfering surfaces. Although previous spectral subtraction was needed in some cases, all the smudged were linked to their corresponding lipsticks even when they are smeared on the interfering surfaces. As a consequence, confocal Raman microscopy using the 780 nm excitation laser is presented as a nondestructive powerful tool for the identification of these tricky samples.