Raman Imaging Research Articles

Biochemistry and Nanomechanical Properties of Human Colon Cells upon Simvastatin, Lovastatin, and Mevastatin Supplementations: Raman Imaging and AFM Studies.

One of the most important areas of medical science is oncology, which is responsible for both the diagnostics and treatment of cancer diseases. Over the years, there has been an intensive development of cancer diagnostics and treatment. This paper shows the comparison of normal (CCD-18Co) and cancerous (CaCo-2) cell lines of the human gastrointestinal tract on the basis of nanomechanical and biochemical properties to obtain information on cancer biomarkers useful in oncological diagnostics. The research techniques used were Raman spectroscopy and imaging and atomic force microscopy (AFM). In addition, the studies also included the effect of the statin compounds—mevastatin, lovastatin, and simvastatin—and their influence on biochemical and nanomechanical changes of cell properties using Raman imaging and AFM techniques. The cytotoxicity of statins was determined using XTT tests.

Simulated fine-needle aspiration diagnosis of follicular thyroid nodules by hyperspectral Raman microscopy and chemometric analysis.

.Significance: Follicular thyroid carcinoma carries a substantially poor prognosis due to its unique biological behavior and less favorable outcomes. In particular, fine-needle aspiration (FNA) biopsies, which play a key role in screening thyroid nodules, cannot differentiate benign from malignant follicular neoplasm.Aim: We report on the use of hyperspectral Raman microscopy in combination with chemometric analysis for identifying and classifying single cells obtained from clinical samples of human follicular thyroid neoplasms.Approach: We used a method intended to simulate the FNA procedure to obtain single cells from thyroid nodules. A total of 392 hyperspectral Raman images of single cells from follicular thyroid neoplasms were collected.Results: Malignant cells were identified based on their intrinsic Raman spectral signatures with an overall diagnostic accuracy of up to 83.7%.Conclusions: Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an ancillary test for analyzing single cells from thyroid FNA biopsies to better stratify “indeterminate” nodules and other cytologically challenging cases.

Raman Spectroscopic Tools to Probe the Skin-(Trans)dermal Formulation Interface.

Medicines designed to deliver the active pharmaceutical ingredient either into or through the skin─often referred to as topicals and transdermals, respectively─are generally considered to be complex drug products. A particular challenge faced by these formulations is identifying a suitable method (ideally, in terms of specificity, accuracy, precision, and robustness) or combination of methods with which to assess the amount and rate of drug delivery to the target site. Significant research currently aims to identify and validate relevant and minimally invasive techniques that can be used to quantify both the levels of the drug attained within different parts of the skin and the kinetics with which the drug is taken up into the skin and cleared therefrom into the systemic circulation. Here, the application of confocal Raman microspectroscopy and imaging to interrogate events integral to the performance of topical and transdermal drug products at the formulation-skin interface is illustrated. Visualization, depth slicing, and profiling are used (a) to elucidate key chemical properties of both the delivery system and the skin that have impact on their interaction and the manner in which drug transfer from one to the other may occur, (b) for the transformation of a drug product from that manufactured into a residual phase post-application and inunction into the skin (including the potential for important changes in solubility of the active compound), and (c) for drug absorption into the skin and its subsequent '"clearance" into deeper layers and beyond. Overall, the Raman tools described offer both qualitative and potentially semi-quantitative insights into topical and transdermal drug product performance and provide information useful for formulation improvement and optimization.

Controlled release of gallium maltolate complex from injectable phosphocalcic cements

Some cancers have tropism for bone: breast, prostate, lung, kidney, and thyroid cancers are the most common. Bone metastases can be treated with surgical resection and the resulting bone defects can be filled with injectable biomaterials. Among these, calcium phosphates may be the biomaterials of choice because of their ability to locally release anticancer active ingredients. Herein, we propose the synthesis of injectable calcium phosphate cement (CPC) loaded with gallium maltolate (GaM). It is an extremely promising anticancer drug with also antibiotic and anti-inflammatory properties. This synthesis was based on commercial cement whose main component was α-tri-calcium phosphate (α-TCP), and the final product obtained after hardening was calcium-deficient apatite (CDA). Two formulations were prepared, containing 3.5% and 7% by mass of GaM (CPC-3.5G and CPC-7G respectively). Powder x-ray diffraction (pXRD), Fourier transform infrared (FTIR) spectroscopy, and magic-angle spinning nuclear magnetic resonance (NMR MAS) 31P analyses showed that the direct incorporation of GaM did not modify the final cement composition. Textural properties, such as setting time, injectability, workability, and cohesiveness, were well preserved or even improved. Additionally, the mechanical strength, although slightly reduced, remained perfectly compatible with surgical use. In vitro kinetics studies of GaM-loaded CPCs showed a controlled release of GaM (49% at 60 days for CPC-3.5G and 58% at 116 days for CPC-7G) following Fick’s law. Raman imaging was used to visualize its diffusion within the cement during in vitro release experiments. Finally, the structural integrity of the gallium complex in the CPC was confirmed using NMR MAS 71Ga.

Elucidating cell surface glycan imbalance through SERS guided metabolic glycan labelling: An appraisal of metastatic potential in cancer cells.

The intrinsic complexities of cell-surface glycans impede tracking the metabolic changes in cells. By coupling metabolic glycan labelling (MGL) and surface-enhanced Raman scattering (SERS), we employed the MGL-SERS strategy to elucidate the differential glycosylation pattern in cancer cell lines. Herein, for the first time, we are reporting an N-alkyl derivative of glucosamine (GlcNPhAlk) as a glycan labelling precursor. The extent of labelling was assessed by utilizing Raman imaging and verified by complementary fluorescence and Western blot analysis. MGL-SERS technique was implemented for a comparative evaluation of cell surface glycan imbalance in different cancer cells wherein a linear relationship between glycan expression and metastatic potential was established. Further, the effect of sialyltransferase inhibitor, P-3Fax-Neu5Ac, on metabolic labelling of GlcNPhAlk proved the incorporation of GlcNPhAlk to the terminal glycans through the sialic acid biosynthetic pathway. Hence, this methodology unveils the phenomenon of metastatic progression in cancer cells with inherent glycosylation-related dysplasia.

Imaging with Raman photons: a novel use of mixed-mode spectroscopy

Surface enhanced Raman spectroscopy is today an established technique used for chemical fingerprinting. Here, we showcase an engineered hierarchical substrate, in which the plasmonically active regions, restricted to a micron scale, two dimensional hexagonal pattern are examined. Spatial variation of the enhanced Raman signal from any analyte, uniformly coating the substrate, consequently bears a high registry with the underlying pattern. This spatially contrasted enhancement allows optical imaging of the 2D pattern solely using the Raman scattered photons from the analyte. While the pattern brightness and contrast determine analyte identification and detection sensitivity, hyperspectral imaging can be exploited for increasing specificity. Proof of concept demonstration of the technique is carried out via the acquisition of Raman images with rhodamine and fluorescein dyes and then applied to detect glucose in 40 mM concentration. The large area optical imaging and the requirement of long-range uniformity in the detected patterns for positive analyte detection, is implemented using a machine learning based pattern recognition protocol which also increases the statistical confidence of detection. This simultaneous, large area signal detection sacrifices continuous spectral information at the cost of speed, reproducibility and minimising human error via automation of detection in the hyperspectral imaging technique presented here.

Focus on lasers, imaging, microscopy, and photonics

Focus on lasers, imaging, microscopy, and photonics

Fire releases micro- and nanoplastics: Raman imaging on burned disposable gloves

Raman imaging can effectively characterise microplastics and nanoplastics, which is validated here to capture the items released from the plastic gloves when subjected to a mimicked fire. During the COVID-19 pandemic, large quantities of personal protective equipment (PPE) units have been used, such as the disposable gloves. If discarded and poorly managed, plastics gloves might break down to release secondary contaminants. The breakdown process can be accelerated by burning in a bushfire or at the incineration plants. During the burning process, the functional groups on the surface can be burned differently due to their different thermal stabilities. The different degrees of burning can be distinguished and visualised via Raman imaging. In the meantime, at the bottom of the burned plastics, microplastics and nanoplastics can be generated at a significant amount. The possible false Raman imaging on microplastics and nanoplastics is also discussed, by effectively extracting and distinguishing the weak signal from the background or noise. Overall, these findings confirm the importance of effectively working waste incineration plants and litter prevention, and suggest that Raman imaging is a suitable approach to characterise microplastics and nanoplastics.

Temperature-mediated construction of a plantar pressure-relieving, thermally insulating, and biodegradable thick-walled cellulose sponge insole

Plantar pressure can be reduced and wearing comfort can be improved by using cushioning materials such as insoles. However, the biodegradation of most commercial insoles is difficult, incurring high disposal costs both economically and environmentally. Herein, we present a temperature-mediated approach for constructing a plantar pressure reducing, thermally insulating, and biodegradable thick-walled cellulose sponge (TWCS) insole from cotton linters. 3D Raman imaging and crystallographic test results demonstrated that freezing temperature affected the packing behavior of the dispersed cellulose at the ice crystal boundary, resulting in a marked distinction between wall thicknesses and crystallinity of sponges, from 0.36 μm and 0.39 to 7.74 μm and 0.46, at distinguishing temperatures (−25 ˚C, −78 ˚C, and − 198 ˚C). Benefiting from the thick-walled structure generated during the temperature-mediated interfacial assembly, the mechanical strength of the TWCS was significantly increased by 5.6 times to 569 kPa, accompanied by significantly enhanced structural stability, pressure-relieving, and thermal insulating characteristics. Subsequently, we designed TWCS as a biomass insole, and its performance was thoroughly examined using plantar pressure testing, thermal performance assessment, finite element simulation, and degradability investigations. The results demonstrate that TWCS can effectively decompress (1-fold pressure reduction), disperse external stresses, prevent heat loss and complete degradation within 50 days under biological conditions, and can potentially replace traditional petroleum-based sponge insoles.

Raman imaging for the identification of Teflon microplastics and nanoplastics released from non-stick cookware

The characterisation of microplastics is still difficult, and the challenge is even greater for nanoplastics. A possible source of these particles is the scratched surface of a non-stick cooking pot that is mainly coated with Teflon. Herein we employ Raman imaging to scan the surfaces of different non-stick pots and collect spectra as spectrum matrices, akin to a hyperspectral imaging process. We adjust and optimise different algorithms and create a new hybrid algorithm to extract the extremely weak signal of Teflon microplastics and particularly nanoplastics. We use multiple characteristic peaks of Teflon to create several images, and merge them to one, using a logic-based algorithm (i), in order to cross-check them and to increase the signal-noise ratio. To differentiate the varied peak heights towards image merging, an algebra-based algorithm (ii) is developed to process different images with weighting factors. To map the images via the whole set of the spectrum (not just from the individual characteristic peaks), a principal component analysis (PCA)-based algorithm (iii) is employed to orthogonally decode the spectrum matrix to the PCA spectrum and PCA intensity image. To effectively extract the Teflon spectrum information, a new hybrid algorithm is developed to justify the PCA spectra and merge the PCA intensity images with the algebra-based algorithm (PCA/algebra-based algorithm) (iv). Based on these developments and with the help of SEM, we estimate that thousands to millions of Teflon microplastics and nanoplastics might be released during a mimic cooking process. Overall, it is recommended that Raman imaging, along with the signal recognition algorithms, be combined with SEM to characterise and quantify microplastics and nanoplastics.

Transdermal diffusion of resveratrol by multilamellar liposomes: Effect of encapsulation on its stability

Trans-Resveratrol (RV) was encapsulated in multi-lamellar liposomes (MLLs) composed of a mixture of phosphatidylcholine (P100), Tween 80 (T80) and water. The P100-to-T80 ratio as well as their water content were chosen based on a previous study to promote transdermal transport of RV. The effect of RV on the size, elasticity, and charge of MLLs was evaluated as well as the effect of encapsulation on the apparent solubility of RV. The diffusion of RV and MLLs in artificial skin, namely Strat-M™, was monitored by confocal Raman imaging, and compared with that of a RV solution and empty MLLs, while the transdermal passage rate was measured by UV–vis spectrophotometry on both artificial and excised human skin. RV was found to remain localized in the outer layer of the skin with less than 3% passing through it over a 24-h period in both skin types. Encapsulation in MLLs drastically increased its transdermal passage: 73 ± 10% after 3 h of incubation on excised human skin, and 10% on Strat-M™ after 9 h. Whereas RV in its free form underwent cis isomerization, MLLs protected it up to 9h before undergoing chemical degradation after 24h.

Coherent anti-Stokes Raman scattering cell imaging and segmentation with unsupervised data analysis.

Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.

3D (x-y-t) Raman imaging of tomato fruit cuticle: Microchemistry during development.

The cuticle is a protective extracellular matrix that covers the above-ground epidermis of land plants. Here, we studied the cuticle of tomato (Solanum lycopersicum L.) fruits in situ using confocal Raman microscopy. Microsections from cuticles isolated at different developmental stages were scanned to visualize cuticle components with a spatial resolution of 342 nm by univariate and multivariate data analysis. Three main components, cutin, polysaccharides, and aromatics, were identified, with the latter exhibiting the strongest Raman scattering intensity. Phenolic acids and flavonoids were differentiated within the cuticle, and three schematic cuticle models were identified during development. Phenolic acids were found across the entire cuticle at the earliest stage of development, i.e. during the formation of the procuticle layer. Based on a mixture analysis with reference component spectra, the phenolic acids were identified as mainly esterified p-coumaric acid together with free p-hydroxybenzoic acid. During the cell expansion period of growth, phenolic acids accumulated in an outermost layer of the cuticle and in the middle region of the pegs. In these stages of development, cellulose and pectin were detected next to the inner cuticle region, close to the epidermal cell where flavonoid impregnation started during ripening. In the first ripening stage, chalconaringenin was observed, while methoxylated chalcones were chosen by the algorithm to fit the mature cuticle spectra. The colocation of carbohydrates, esterified p-coumaric acid, and methoxylated chalconaringenin suggests that the latter two link polysaccharide and cutin domains. Elucidating the different distribution of aromatics within the cuticle, suggests important functions: (1) overall impregnation conferring mechanical and thermal functions (2) the outermost phenolic acid layer displaying UV-B protection of the plant tissue.

High-Resolution Low-Power Hyperspectral Line-Scan Imaging of Fast Cellular Dynamics Using Azo-Enhanced Raman Scattering Probes.

Small-molecule Raman probes for cellular imaging have attracted great attention owing to their sharp peaks that are sensitive to environmental changes. The small cross section of molecular Raman scattering limits dynamic cellular Raman imaging to expensive and complex coherent approaches that acquire single-channel images and lose hyperspectral Raman information. We introduce a new method, dynamic azo-enhanced Raman imaging (DAERI), to couple the new class of azo-enhanced Raman probes with a high-speed line-scan Raman imaging system. DAERI achieved high-resolution low-power imaging of fast cellular dynamics resolved at ∼270 nm along the confocal direction, 75 μW/μm2 and 3.5 s/frame. Based on the azo-enhanced Raman probes with characteristic signals 102-104 stronger than classic Raman labels, DAERI was not restricted to the cellular Raman-silent region as in prior work and enabled multiplex visualization of organelle motions and interactions. We anticipate DAERI to be a powerful tool for future studies in biophysics and cell biology.

Comparison of protective coatings prepared from various trialkoxysilanes and possibilities of spectroelectrochemical approaches for their investigation

Abstract Herein protective coatings against corrosion are compared with regard to their protective efficiency. The coatings had been prepared over the course of time from differently trialkoxysilyl-functionalized commercial or synthesized precursors but were tested using the same techniques and in similar conditions. The coatings are compared according to the existing data on contact angles for water, free surface energy, thickness and protective efficiency, expressed in term of corrosion current density. Moreover, the influence of differently functionalized trialkoxysilyl precursors on the crosslinking of coatings is regarded. It was also noted that too hydrophobic additives can introduce certain defects which can detrimentally influence the protective efficiency. Spectroelectrochemical approach can give important insights into the degradation of protective coatings under electrochemically induced loads. Ex situ infrared reflection-absorption (IR RA) spectroelectrochemical approach can identify hydration, breakage of some siloxane bands or changes in the C–H spectral region. Careful examination can confirm the interruption of some bands between alloy and coating that are responsible for its adhesion. Raman imaging is appropriate to follow the formation and growth of pits that form in the coatings.

Super tough interpenetrating polymeric network of styrene butadiene rubber‐poly(methyl methacrylate) incorporated with general purpose carbon black (N660)

AbstractA classic set of polymeric interpenetrating polymeric network (IPN) micro‐composites has been fabricated using an elastomer—styrene butadiene rubber [SBR], a thermoplastic poly(methyl methacrylate)‐PMMA and with carbon black (CB)‐N660 as a filler and reinforcing agent. This synthesized IPN composite can be promisingly employed as a toughened plastic and vibrational damper in a wide service range with excellent thermal stability, optimum storage modulus, and co‐continuous morphological pattern. transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and Raman imaging are successfully employed for the morphological characterization. Mechanical, thermal, viscoelastic, and damping features of IPN composites have been carefully studied in detail and compared with parent polymers, corresponding IPN, and composites. The double network formation of filler CB and plastic component PMMA form an intercalated morphological pattern in the SBR matrix with 20 times enhancement in toughness value compared with neat SBR. The fabrication and characterization adopted in this work can definitely act as a platform for the design of new toughened material with excellent performance and cost‐effectiveness.

Rapid Limit Test of Seven Pesticide Residues in Tea Based on the Combination of TLC and Raman Imaging Microscopy

Numerous studies have shown that pesticide residues in tea exceeding the maximum residue limits (MRL) can cause harmful effects on the human body. There are many limitations in the existing analytical methods for pesticide residues in tea, so new analytical methods need to be developed. We developed a limit test method that combines thin-layer chromatography with Raman imaging microscopy (TLC-RIM). Seven residual pesticide components in tea (Avermectin, Methomyl, Carbendazim, Imidacloprid, Chlorothalonil, Azoxystrobin, and Acetamiprid) could be preliminarily separated by TLC and then irradiated by a 532 nm laser. Raman spectra of seven pesticides obtained by Raman imaging microscopy could be used to test whether the pesticide residues in tea exceed the MRL. The limits of detection of the seven pesticides were 0.04, 0.10, 0.24, 0.20, 0.12, 0.12, and 1.0 mg/mL, respectively. The simulated positive test showed that the matrix in tea did not interfere with the test of the seven pesticides. When the pesticides were tested within 8 h, the RSD of the peak heights of the seven pesticides were 1.2%~9.6%; the test results of three batches of tea showed that the imidacloprid in one batch of tea exceeded its MRL, and the results were consistent with that by UPLC-MS/MS. The TLC-RIM is fast, sensitive, stable, specific, and reliable.

Labelfree mapping and profiling of altered lipid homeostasis in the rat hippocampus after traumatic stress: Role of oxidative homeostasis

Oxidative and lipid homeostasis are altered by stress and trauma and post-traumatic stress disorder (PTSD) is associated with alterations to lipid species in plasma. Stress-induced alterations to lipid and oxidative homeostasis may exacerbate PTSD pathology, but few preclinical investigations of stress-induced lipidomic changes in the brain exist. Currently available techniques for the quantification of lipid species in biological samples require tissue extraction and are limited in their ability to retrieve spatial information. Raman imaging can overcome this limitation through the quantification of lipid species in situ in minimally processed tissue slices. Here, we utilized a predator exposure and psychosocial stress (PE/PSS) model of traumatic stress to standardize Raman imaging of lipid species in the hippocampus using LC-MS based lipidomics and these data were confirmed with qRT-PCR measures of mRNA expression of relevant enzymes and transporters. Electron Paramagnetic Resonance Spectroscopy (EPR) was used to measure free radical production and an MDA assay to measure oxidized polyunsaturated fatty acids. We observed that PE/PSS is associated with increased cholesterol, altered lipid concentrations, increased free radical production and reduced oxidized polyunsaturated fats (PUFAs) in the hippocampus (HPC), indicating shifts in lipid and oxidative homeostasis in the HPC after traumatic stress.

EdU sensing: The Raman way of following endothelial cell proliferation in vitro and ex vivo

Endothelial cells line the lumen of all vessels in the body and maintain vascular homeostasis. In particular, endothelial cell regeneration in response to insult sustain functional endothelial layer. EdU (5-ethynyl-2′-deoxyuridine) is an alkyne-tagged proliferation probe that incorporates into newly synthesized DNA and is used for fluorescence imaging of cell proliferation with the use of “click chemistry” reaction with a fluorescent azide. Here, we utilized EdU as a click-free Raman probe for tracking endothelial cell proliferation. Raman imaging of EdU was performed in live endothelial cells, showing an advantage over fluorescence imaging of EdU, as this technique did not require sample fixation and permeabilization. To validate Raman-based imaging of EdU to study endothelial cell proliferation, we showed that when endothelial cells were treated with cycloheximide or doxorubicin to impair the proliferation of endothelial cells, the Raman-based signal of EdU was diminished. Furthermore, endothelial cells proliferation detected using EdU-labelled Raman imaging was compared with fluorescence imaging. Finally, the method of Raman–based EdU imaging was used in the isolated murine aorta ex vivo. Altogether, our results show that Raman-based imaging of EdU provides a novel alternative for fluorescence-based assay to assess endothelial proliferation and regeneration.

Design, Synthesis, and Analytical Evaluation of Fsp3‐Inspired Raman Probes for Cellular Imaging

AbstractThe fraction of sp3‐hybdrised carbons (Fsp3) in drug candidates and other biologically relevant compounds has been proposed to be an important physicochemical consideration within medicinal chemistry. Currently available small‐molecule probes for Raman imaging are not optimised for biological applications. We sought to improve upon the highly Raman‐active, yet rigid, lipophilic, and aggregative, bisarylbutadiyne (BADY) motif by introducing Fsp3 character. BADY analogues were designed, efficiently synthesised, and analysed by mass spectrometry – revealing a statistical correlation between compound aggregation and cLogP, but no correlation with Fsp3. However, X‐ray crystallography demonstrated that the modifications to the BADY structure had a marked effect on crystal packing, suggesting Fsp3 may still be important in reducing intermolecular stacking, and thus aggregation, of BADY. Cellular imaging by stimulated Raman scattering (SRS) microscopy allowed comparison of the intracellular distribution of the tags, guiding the design of future Raman probes.