Raman Imaging Research Articles

Surface‐enhanced Raman imaging of intact cancer cell membrane on a rough aluminum substrate

AbstractThe plasma membrane of cancer cells displays differences in lipid and protein composition which distinguish them from healthy cells. Certain molecules have such prominent vibrational characteristics that they can be considered cancer biomarkers. Although Raman spectroscopy is a label‐free non‐invasive method of chemical analysis, it, however, has been limited by weak signals from the membrane compared to the bulk interior of cells. Surface‐enhanced Raman scattering (SERS) on plasmonic gold and silver substrates has previously been used to overcome this limitation. However, the high cost, poor repeatability, and toxicity of these noble metals hinder their practical applications. These challenges may be solved by using different materials and nanostructures. Here, we performed SERS imaging of ovarian cancer cells on rough aluminum substrates and extracted the Raman signals of intact cell membranes by comparing these with the corresponding measurements on Si substrates. We used atomic force microscopy (AFM) for Raman signal calibration. The comparison of cancer and normal cell membranes revealed the different lipid/protein peak ratios in cytoplasm and nucleus‐proximal areas. These Raman signatures of the ovarian cancer can be used as new biomarkers. This method may be extended to other types of cancer biosensing based on aluminum plasmonics.

Area-Selective Depolymerization of Hydroxycinnamates Visualized by Raman Imaging in Miscanthus sinensis cv. Cell Wall.

Lignin-carbohydrate complexes (LCCs) are regarded as a barrier for lignocellulosic biomass refinery. Here, confocal Raman microspectroscopy has been used to visualize the dissolution of hydroxycinnamates (HCMs) incorporated into LCCs by ether and ester bonds for energy crops Miscanthus sinensis cv. during successive NaOH (2.5% w/w) treatment. Raman spectral analysis indicated that mild NaOH treatment resulted in a higher proportion of HCM depolymerization in highly lignified middle lamella areas (>66.0%) than carbohydrate-abundant secondary walls. Furthermore, Raman imaging revealed preferential depolymerization of lignin from the sclerenchyma fiber (Sf) and parenchyma (Par) secondary wall with an increment of treatment time from 0 to 25 min, while middle lamella areas of Sf and Par were less affected where the depolymerization of HCMs was closely related with that of lignin (coefficient factors > 0.96). A better understanding of the depolymerization behavior for HCMs accompanied by lignin depolymerization was important to break LCC bonds in herbaceous biomass efficiently.

Toward Gene-Correlated Spatially Resolved Metabolomics with Fingerprint Coherent Raman Imaging.

Raman spectroscopy has long been known to provide sufficient information to discriminate distinct cell phenotypes. Underlying this discriminating capability is that Raman spectra provide an overall readout of the metabolic profiles that change with transcriptomic activity. Robustly associating Raman spectral changes with the regulation of specific signaling pathways may be possible, but the spectral signals of interest may be weak and vary somewhat among individuals. Establishing a Raman-to-transcriptome mapping will thus require tightly controlled and easily manipulated biological systems and high-throughput spectral acquisition. We attempt to meet these requirements using broadband coherent anti-Stokes Raman scattering (BCARS) microscopy to spatio-spectrally map the C.elegans hermaphrodite gonad in vivo at subcellular resolution. The C.elegans hermaphrodite gonad is an ideal model system with a sequential, continuous process of highly regulated spatiotemporal cellular events. We demonstrate that the BCARS spatio-spectral signatures correlate with gene expression profiles in the gonad, evincing that BCARS has potential as a spatially resolved omics surrogate.

Improved Reliability of Raman Spectroscopic Imaging of Low-Micrometer Microplastic Mixtures in Lake Water by Fractionated Membrane Filtration

Low-micrometer microplastics (LMMPs), typically in the size range, 1–10 μm, are emerging contaminants ubiquitously present in aquatic systems. However, the quantification of LMMP mixtures in environmental waters is hindered by the limitations of current analytical methods. In this study, we developed a novel analytical method that couples a fractionated membrane filtration with Raman microspectroscopy (μ-Raman) to reliably quantify LMMPs in lake water. The fractionated membrane filtration system recovered >90% of the mixed polystyrene (PS) and poly(methyl methacrylate) (PMMA) LMMPs (i.e., 1.5, 3, 5, and 8 μm) from water. By separating LMMPs into 1–5 and 5–10 μm size fractions, the quality of the Raman images of small LMMPs (i.e., 1.5 and 3 μm particles) acquired directly on the membrane filters improved substantially. The reliability of LMMP Raman imaging was further improved by tracking the sum and product of their three characteristic Raman bands. In this way, the interferent Raman signals from the lake water matrix and membrane filters were significantly suppressed. This method provides a reliable tool to recover, separate, and quantify LMMP mixtures in complex water matrices, paving the way toward a deeper understanding of their environmental implications.

Effect of defects on Q factors of single-crystal diamond MEMS resonators

A resonator with a high Q factor is generally pursued in the single-crystal diamond (SCD) microelectromechanical system (MEMS) for high-performance sensors. In this report, we investigate the oxygen etching effect of SCD on the Q factors of the SCD resonators by using the Raman spectroscopy spatial mapping. We aim to establish the etch pit effect on the Q factors of the SCD MEMS resonators. The 2D Raman imaging technique discloses the dislocations and the local stress in the SCD MEMS resonators in microscale. It is observed that the full width half maximum (FWHM) of the Raman spectra of the SCD resonators has marked relationship with the Q factors of the SCD resonators. The etch pits resulted from the dislocations have weak influence on the Q factors of the SCD resonators.

Chemical composition changes of kiwifruit petiole cell walls infected by Pseudomonas syringae pv. actinidiae based on confocal Raman imaging combined with chemometrics

Kiwifruit canker caused by Pseudomonas syringae pv. actinidiae (Psa) is devastating to the kiwifruit industry. Because of the complex pathogenesis of kiwifruit canker, there is still a lack of effective control measures. The aim of this study was to characterize the temporal and spatial changes of biopolymers in the petiole cell walls of healthy and cankered kiwifruit using confocal Raman microscopy (CRM) techniques combined with Chemometrics. We proposed a general investigation of the development of kiwifruit canker and chemical distribution imaging was successfully applied to the analysis of disease dynamics. First, Raman spectra (RS) of healthy and diseased kiwifruit petiole cell walls were acquired at a micron-scale spatial resolution, followed by label-free in situ imaging of the cell wall. The results of RS and chemical imaging showed that the changes of cellulose and lignin on the petiole cell wall of susceptible kiwifruit under Psa stress were time-dependent, and the degradation rate of cellulose and lignin reached about 25% after eight days of incubation. The results of principal component analysis (PCA) and support vector machine (SVM) based on RS showed that healthy, early-stage disease and late-stage disease samples exhibited significant clustering effects and the SVM classification accuracy was as high as 100%. The multivariate curve resolved–alternating least squares (MCR–ALS) is used to optimize the Raman imaging in order to eliminate the noise and interference caused by the peak clamping method and provide a meaningful chemical profile. In conclusion, CRM combined with chemical classification algorithms and imaging analysis has certain feasibility and application prospects in the early warning and cellular-level dynamic characterization of kiwifruit canker.

Label-Free Evaluation of Maturation and Hepatotoxicity of Human iPSC-Derived Hepatocytes Using Hyperspectral Raman Imaging.

To promote the clinical application of human induced pluripotent stem cell (hiPSC)-derived hepatocytes, a method capable of monitoring regenerative processes and assessing differentiation efficiency without harming or modifying these cells is important. Raman microscopy provides a powerful tool for this as it enables label-free identification of intracellular biomolecules in live samples. Here, we used label-free Raman microscopy to assess hiPSC differentiation into hepatocyte lineage based on the intracellular chemical content. We contrasted these data with similar phenotypes from the HepaRG and from commercially available hiPSC-derived hepatocytes (iCell hepatocytes). We detected hepatic cytochromes, lipids, and glycogen in hiPSC-derived hepatocyte-like cells (HLCs) but not biliary-like cells (BLCs), indicating intrinsic differences in biomolecular content between these phenotypes. The data show significant glycogen and lipid accumulation as early as the definitive endoderm transition. Additionally, we explored the use of Raman imaging as a hepatotoxicity assay for the HepaRG and iCell hepatocytes, with data displaying a dose-dependent reduction of glycogen accumulation in response to acetaminophen. These findings show that the nondestructive and high-content nature of Raman imaging provides a promising tool for both quality control of hiPSC-derived hepatocytes and hepatotoxicity screening.

Analytical Techniques for Single-Cell Biochemical Assays of Lipids.

Lipids are essential cellular components forming membranes, serving as energy reserves, and acting as chemical messengers. Dysfunction in lipid metabolism and signaling is associated with a wide range of diseases including cancer and autoimmunity. Heterogeneity in cell behavior including lipid signaling is increasingly recognized as a driver of disease and drug resistance. This diversity in cellular responses as well as the roles of lipids in health and disease drive the need to quantify lipids within single cells. Single-cell lipid assays are challenging due to the small size of cells (∼1 pL) and the large numbers of lipid species present at concentrations spanning orders of magnitude. A growing number of methodologies enable assay of large numbers of lipid analytes, perform high-resolution spatial measurements, or permit highly sensitive lipid assays in single cells. Covered in this review are mass spectrometry, Raman imaging, and fluorescence-based assays including microscopy and microseparations.

Insights into S. aureus-Induced Bone Deformation in a Mouse Model of Chronic Osteomyelitis Using Fluorescence and Raman Imaging.

Osteomyelitis is an infection of the bone that is often difficult to treat and causes a significant healthcare burden. Staphylococcus aureus is the most common pathogen causing osteomyelitis. Osteomyelitis mouse models have been established to gain further insights into the pathogenesis and host response. Here, we use an established S. aureus hematogenous osteomyelitis mouse model to investigate morphological tissue changes and bacterial localization in chronic osteomyelitis with a focus on the pelvis. X-ray imaging was performed to follow the disease progression. Six weeks post infection, when osteomyelitis had manifested itself with a macroscopically visible bone deformation in the pelvis, we used two orthogonal methods, namely fluorescence imaging and label-free Raman spectroscopy, to characterise tissue changes on a microscopic scale and to localise bacteria in different tissue regions. Hematoxylin and eosin as well as Gram staining were performed as a reference method. We could detect all signs of a chronically florid tissue infection with osseous and soft tissue changes as well as with different inflammatory infiltrate patterns. Large lesions dominated in the investigated tissue samples. Bacteria were found to form abscesses and were distributed in high numbers in the lesion, where they could occasionally also be detected intracellularly. In addition, bacteria were found in lower numbers in surrounding muscle tissue and even in lower numbers in trabecular bone tissue. The Raman spectroscopic imaging revealed a metabolic state of the bacteria with reduced activity in agreement with small cell variants found in other studies. In conclusion, we present novel optical methods to characterise bone infections, including inflammatory host tissue reactions and bacterial adaptation.

The reconstruction of serial numbers in polymers: Recent progress, challenges, and perspectives

AbstractThe mass production of polymers has forced forensic practitioners to reconsider traditional methods of traces analysis. The field of impression reconstruction, specifically markings in firearms, is a prime example. This overview offers a critical evaluation of the relevant published techniques for the reconstruction of serial number in polymers, which include destructive methods such as swelling, heat treatments and relief polishing as well as methods allowing for trace preservation, such as hyperspectral Raman imaging combined with multivariate statistical analysis for enhanced pertinent data extraction. It therefore provides a complementary compilation to existing protocols for metal substrates. The novelty of this work lies within its approach, specifically by establishing not only the mechanistic scientific explanation for suitable comprehension and application of the techniques, but also by properly assessing their relevance considering the use in a forensic science context. The potential of wide‐field imaging techniques, mainly auto‐fluorescence analysis, is suggested for faster acquisition and reduced data processing (i.e., decreased time and greater accessibility). Additionally, application of the acquired knowledge to other relevant forensic traces, such as failure analysis of 3D printed objects, is proposed. Emphasis is also placed on the relevance of a purposeful interpretative framework necessary to reconstruct the singular past of the obliterated serial number thus leading to the identification of the given object in which it is affixed.This article is categorized under: Forensic Chemistry and Trace Evidence > Fingermarks and Other Marks Forensic Chemistry and Trace Evidence > Emerging Technologies and Methods Forensic Chemistry and Trace Evidence > Trace Evidence

Graphene-coated nylon nonwoven for pressure sensing application

Graphene-coated textiles have attracted great interest in recent years for the development of advanced piezo-resistive materials. In this work, rGO (reduced graphene oxide) treated nylon nonwoven is prepared by the dip coating method. The influence of treatment parameters viz. graphene oxide (GO) concentration, sodium dithionite (Na2S2O4) concentration, and, pH of GO dispersion on rGO add-on and electrical conductivity is analysed and optimized by using Box–Behnken response surface design to prepare electroconductive nylon nonwoven for pressure sensing application. An rGO add-on of 9.12% and surface resistivity of 3.62 kΩ sq−1 have been achieved after two dippings at the optimized dipping condition viz. 0.6% GO concentration, 2 pH, and 15 mM Na2S2O4 concentration. Surface resistivity of 3.62 kΩ sq −1 is achieved after two dippings at the optimized dipping condition with the rGO add-on of 9.12%. The piezoresistive properties of this fabric are investigated. Resistance and thickness of rGO-coated nylon nonwoven treated fabric show a strong negative correlation with the applied pressure. A maximum gauge factor of 2.56 is observed which is in line with pure compression-based pressure sensors. A high-pressure sensitivity of − 0.34 kPa −1 is observed at 2 kPa pressure with a recovery time of 1.2 s implying that it can be used for pressure sensing. After rGO dipping, bending rigidity, bending modulus, and breaking strength of nylon nonwoven are increased whereas the breaking elongation decreased. Bending rigidity, bending modulus, and breaking strength of nylon nonwoven increase after rGO dipping, while breaking elongation decreases. Surface resistivity and rGO add-on of treated nylon nonwoven remain unchanged even after multiple washing, rubbing, and atmospheric ageing. Nylon nonwovens are characterised by FT-IR, XRD, Raman, FESEM, and LEICA image analyser analysis are used to characterise the developed nonwovens.

Surface-enhanced Raman imaging through sprayed probes for the application in chemical visualization of methamphetamine within fingerprints.

For fingerprint-involved forensic investigations, cyanoacrylates and inorganic nanophosphors are mostly used for fingerprint visualization. However, methods to simultaneously report fingerprint images and the corresponding specific chemical information have yet to be realized. In this work, chemical visualization of the analytes in fingerprints is achieved through surface-enhanced Raman spectroscopy (SERS) measurements with the aid of spray-dispersed gold nanorods (AuNRs). The optimal coverage of AuNRs was studied by theoretical simulations and experimental operations. A rapid sampling of fingerprints with the chemical of interest was developed by tuning the spray parameters. In particular, the SERS imaging of methamphetamine in fingerprint latent was attempted by addressing the SERS spectral features of methamphetamine. This chemical visualization method reflects both the graphical and chemical characteristics of fingerprints in a single batch measurement, in which methamphetamine can be detected and mapped at the concentration of 10-5M. The data processing approach was also modified by employing relevant logical judgments. The improved SERS images with sharpened patterns of fingerprints were obtained by involving the scored multi-peak judgments.

Selective Detection in Impulsive Low-Frequency Raman Imaging Using Shaped Probe Pulses

Impulsive stimulated Raman scattering (ISRS) using a single short femtosecond pump pulse to excite molecular vibrations offers an elegant pump-probe approach to perform vibrational imaging below $200\phantom{\rule{0.2em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$. One shortcoming of ISRS is its inability to offer vibrational selectivity as all the vibrational bonds whose frequencies lie within the short pump-pulse bandwidth are excited. To date, several coherent control techniques have been explored to address this issue and selectively excite a specific molecular vibration by shaping the pump pulse. There has not been any systematic work that reports an analogous shaping of the probe pulse to implement preferential detection. In this work, we focus on vibrational imaging and report vibrational selective detection by shaping the probe pulse in time. We demonstrate numerically and experimentally two pulse-shaping strategies with one functioning as a vibrational notch filter and the other functioning as a vibrational low-pass filter. This enables fast ($25\phantom{\rule{0.2em}{0ex}}\text{\ensuremath{\mu}}\mathrm{s}/$pixel) and selective hyperspectral imaging in the low-frequency regime ($<200\phantom{\rule{0.2em}{0ex}}{\mathrm{cm}}^{\ensuremath{-}1}$).

Chemical tissue heterogeneity of young Arabidopsis stems revealed by Raman imaging combined with multivariate data analysis

Structural and chemical tissue heterogeneity in plant stems is essential to fulfil the many different functions for growth and survival. Cutting microsection of young developing stems of the model plant Arabidopsis opened the view on vascular bundles (transport of water, nutrients, food, and other chemicals), interfascicular fibers (mechanical support), parenchyma (production, storage) and the epidermis with cuticle (protection, barrier, exchange,..). Mapping such a cross-section with a Confocal Raman microscope resulted in hyperspectral datasets, which are the basis to image chemical heterogeneity with a spatial resolution of 300 nm in context with the microstructure. We generated the images based on three different multivariate approaches: unmixing to find the most pure components, cluster analysis so segment the dataset into clusters with spectral similarity, and fitting the original spectra at every pixel by a linear combination of plant component reference spectra. All three visualized chemical heterogeneity and confirmed in a complementary way the distribution of carbohydrates and aromatic components. The true component analysis was superior to cluster analysis in specificity and added information on lipids and starch distribution. Due to the multicomponent nature of plant tissues no “pure” components were retrieved, wherefore a subsequent mixture analysis (orthogonal matching pursuit) of extracted component spectra with a reference database followed. This led to details on the molecular composition of the spectra and tissues and was essential input for a final reference component fit at every pixel. By the last analysis, different aromatic components and hemicelluloses were discriminated and a similarity of their distribution patterns elucidated. Insights into starch and lipid distribution as well as the aromatic and protein co-location (mixtures) of cell contents were gained. The fit of the latter lumen content was inferior to the other fits due to noisier spectra (higher fluorescence background) and relevant protein and enzyme spectra were missing in our database. The gained comprehensive view on plant stem tissue heterogeneity showed the potential for future studies to unravel plant species characteristics and adaptations, but also to follow plant developmental processes and/or stress responses and/or genetic modifications.

Raman imaging for the analysis of silicone microplastics and nanoplastics released from a kitchen sealant.

Plastic products are used ubiquitously and can potentially release microplastics and nanoplastics into the environment, for example, products such as the silicone sealant used in kitchens. It is important to develop an effective method to monitor these emerging contaminants, as reported herein. By using advanced Raman imaging to characterize microplastics and nanoplastics from hundreds of spectra in a scanning spectrum matrix and not from a single spectrum or peak, the signal-to-noise ratio can be significantly increased, from a statistical point of view. The diffraction of the laser spot usually constrains the imaging resolution (such as at ∼300nm), which is also pushed to the limit in this report by shrinking the scanning pixel size down to ∼50nm to capture and image small nanoplastics effectively. To this end, image reconstruction is developed to successfully pick up the meaningful Raman signal and intentionally avoid the noise. The results indicate that the silicone sealant in a kitchen can release a significant amount of microplastics and nanoplastics. Overall, advanced Raman imaging can be employed to characterize the microplastics and even nanoplastics that are smaller than the diffraction limit of the laser via Raman imaging and image reconstruction toward deconvolution.

Stretching the Bisalkyne Raman Spectral Palette Reveals a New Electrophilic Covalent Motif.

Small heteroaryl-diyne (Het-DY) tags with distinct vibrational frequencies, and physiologically relevant cLog P were designed for multiplexed bioorthogonal Raman imaging. Pd-Cu catalyzed coupling, combined with the use of Lei ligand, was shown to improve overall yields of the desired heterocoupled Het-DY tags, minimizing the production of homocoupled side-products. Spectral data were in agreement with the trends predicted by DFT calculations and systematic introduction of electron- rich/poor rings stretched the frequency limit of aryl-capped diynes (2209-2243 cm-1 ). The improved Log P of these Het-DY tags was evident from their diffuse distribution in cellular uptake studies and functionalizing tags with organelle markers allowed the acquisition of location-specific biological images. LC-MS- and NMR-based assays showed that some heteroaryl-capped internal alkynes are potential nucleophile traps with structure-dependent reactivity. These biocompatible Het-DY tags, equipped with covalent reactivity, open up new avenues for Raman bioorthogonal imaging.

Layer-dependent stability of 2D mica nanosheets

We report on the layer-dependent stability of muscovite-type two-dimensional (2D) mica nanosheets (KAl3Si3O10(OH)2). First-principles calculations on mica nanosheets with different layer thicknesses (n = 1, 2, and 3) reveal their layer-dependent stability; odd-numbered 2D mica nanosheets are more stable than even-numbered ones, and the preferable stability of odd-numbered layers originates from electronic effects. A core-shielding model is proposed with a reasonable assumption, successfully proving the instability of the even-numbered mica nanosheets. Raman imaging supports that the population of odd-numbered mica nanosheets is predominant in exfoliated mica products. The alternating charge states with odd/even layers were evidenced by Kelvin probe force microscopy. We also demonstrate a unique photocatalytic degradation, opening new doors for environmental applications of mica nanosheets.

Exploring dynamic changes of fungal cellular components during nanoemulsion treatment by multivariate microRaman imaging

Recently, essential oils (EO) have gained a lot of interest for use as antifungal agent in food and agricultural industry and extensive research is ongoing to understand their mode of action. However, the exact mechanism is not yet elucidated. Here, we integrated spectral unmixing and Raman microspectroscopy imaging to unveil the antifungal mechanism of green tea EO based nanoemulsion (NE) against Magnaporthe oryzae. The dramatic change in protein, lipid, adenine, and guanine bands indicate that NE has a significant impact on the protein, lipid and metabolic processes of purine. The results also demonstrated that the NE treatment caused damage to fungal hyphae by inducing a physical injury leading to cell wall damage and loss of integrity. Our study shows that MCR−ALS (Multivariate Curve Resolution-Alternating Least Squares) and N-FINDR (N-finder algorithm) Raman imaging could serve as a suitable complementary package to the traditional methods, for revealing the antifungal mechanism of action of EO/NE.

Multiple Bioimaging Applications Based on the Excellent Properties of Nanodiamond: A Review.

Nanodiamonds (NDs) are emerging as a promising candidate for multimodal bioimaging on account of their optical and spectroscopic properties. NDs are extensively utilized for bioimaging probes due to their defects and admixtures in their crystal lattice. There are many optically active defects presented in NDs called color centers, which are highly photostable, extremely sensitive to bioimaging, and capable of electron leap in the forbidden band; further, they absorb or emit light when leaping, enabling the nanodiamond to fluoresce. Fluorescent imaging plays a significant role in bioscience research, but traditional fluorescent dyes have some drawbacks in physical, optical and toxicity aspects. As a novel fluorescent labeling tool, NDs have become the focus of research in the field of biomarkers in recent years because of their various irreplaceable advantages. This review primarily focuses on the recent application progress of nanodiamonds in the field of bioimaging. In this paper, we will summarize the progress of ND research from the following aspects (including fluorescence imaging, Raman imaging, X-ray imaging, magnetic modulation fluorescence imaging, magnetic resonance imaging, cathodoluminescence imaging, and optical coherence tomography imaging) and expect to supply an outlook contribution for future nanodiamond exploration in bioimaging.

Visualizing drug-induced lipid accumulation in lysosomes of live cancer cells with stimulated Raman imaging.

The low pH of the lysosomal compartment often results in sequestration of chemotherapeutic agents that contain positively charged basic functional groups, leading to anti-cancer drug resistance. To visualize drug localization in lysosomes and its influence on lysosomal functions, we synthesize a group of drug-like compounds that contain both a basic functional group and a bisarylbutadiyne (BADY) group as a Raman probe. With quantitative stimulated Raman scattering (SRS) imaging, we validate that the synthesized lysosomotropic (LT) drug analogs show high lysosomal affinity, which can also serve as a photostable lysosome tracker. We find that long-term retention of the LT compounds in lysosomes leads to the increased amount and colocalization of both lipid droplets (LDs) and lysosomes in SKOV3 cells. With hyperspectral SRS imaging, further studies find that the LDs stuck in lysosomes are more saturated than the LDs staying out of the lysosomes, indicating impaired lysosomal lipid metabolism by the LT compounds. These results demonstrate that SRS imaging of the alkyne-based probes is a promising approach to characterizing the lysosomal sequestration of drugs and its influence on cell functions.