Raman Imaging Research Articles

Modulating the interaction of graphenic substrates with human interleukin-6 and its monoclonal antibody: a study by Raman images.

Interleukin-6 (IL-6) is a cytokine with wide-ranging biological effects, playing an important role on the immune system and inflammatory responses. Therefore, it is important to develop alternative, highly sensitive and reliable analytical methodologies for the accurate detection of this biomarker in biological fluids. Graphene substrates (GS), such as pristine graphene (G), graphene oxide (GO), and reduced graphene oxide (rGO), have shown great benefits for biosensing and in the development of novel biosensor devices. In this work, we present a proof of concept for the development of a new analytical platform for the specific recognition of human interleukin-6, that is based on the coffee-ring formation of monoclonal antibodies of interleukin-6 (mabIL-6) onto amine functionalized GS. The prepared GS/mabIL-6/IL-6 systems were successfully used to show that IL-6 was specifically and selectively adsorbed onto the area of the mabIL-6 coffee-ring. Raman imaging was confirmed as a versatile tool to investigate different antigen-antibody interactions and their surface distribution. This experimental approach can be used to develop a wide variety of substrates for antigen-antibody interaction allowing the specific detection of an analyte in a complex matrix.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Nontargeted detection and recognition of adulterants in milk powder using Raman imaging and neural networks.

Raman imaging technology combined with targeted chemometrics can play a vital role in the rapid detection of milk powder adulteration, which threatens the lives of infants and other people. However, these methods always suffer from a narrow detection range. Nontargeted methods show a broader detection range but cannot recognize adulterants. Here, a novel nontargeted chemometric method, named as the adversarial discrimination neural network (ADNN), is proposed to detect and recognize adulterants simultaneously. The method comprises building a tight boundary in the feature space of Raman images to discriminate milk powder samples from the majority of adulterated cases. Then a first-order partial derivative of the ADNN is calculated to recognize different adulterants through a local approximation strategy. A validation set containing samples adulterated with various adulterants at concentrations ranging from 0.3% to 1.5% w/w was provided to challenge the proposed method. The validated detection accuracy of the proposed method for authentic and adulterated samples was 99.9% and 99.7% and the adulterants were recognized correctly. The ADNN-Raman represents a novel nontargeted and end-to-end tool for detecting and recognizing adulterants in milk powder simultaneously, providing new insights into nontargeted chemometric analysis.

Preparation and Characterization of DNA-assembled GRS-DNA-CuS Nanodandelions.

In this study, we introduce a detailed protocol for the preparation of DNA-assembled GRS-DNA-copper sulfide (CuS) nanodandelion, a multifunctional theranostics nanoparticle. Using transmission electron microscope (TEM) and dynamic light scattering techniques, we characterize the physicochemical property of DNA-assembled GRS-DNA-CuS nanodandelions and their dissociation property after the first near-infrared (NIR) light irradiation. In addition, we systematically monitor the processes of tumor accumulation and uniform intratumoral distribution (UITD) of ultrasmall CuS photothermal agents (PAs), which are dissociated from GRS-DNA-CuS nanodandelions, by Raman imaging and photoacoustic imaging, respectively. The UITD of the dissociated ultrasmall CuS PAs can enhance the therapeutic efficiency of photothermal treatment under the second NIR light irradiation. Overall, this protocol provides a powerful tool to achieve UITD of PAs by explosively breaking the hydrogen bonds of DNA in GRS-DNA-CuS nanodandelions under NIR light irradiation. We expect DNA-assembled nanotheranostics to serve as a robust platform for a variety of biomedical applications related to photothermal therapy in the oncology field. This protocol can increase experimental reproducibility and contribute to efficient theranostics nanomedicine.

3-D Raman Imaging Using Time-Resolving CMOS SPAD Line Sensor and 2-D Mapping

The capability of Raman imaging to produce 2D and 3D chemical presentations of samples has gained a lot of interest in different application fields. In this paper, we present a 3D chemical image reconstruction based on 2D scanning of a sample utilizing a time-resolved Raman spectrometer based on a CMOS single-photon avalanche diode (SPAD) line sensor. The 2D scanning data contains the lateral information (XY-plane) whereas the time-of-arrival data of the Raman photons measured by the sensor carries the axial information (i.e., depth information, Z-axis). The sensor is fabricated in 110-nm CMOS technology. It has 256-spectral channels and each channel has its own 7-bit on-chip time-to-digital converter (TDC) with an adjustable resolution from 25 ps to 65 ps. In addition to the 3D chemical reconstruction of the scanned sample, we have also shown the ability to retrieve depth profiling information of each scanned pixel such as the boundaries and middle points of any selected layer over the depth range of the scanned object by means of a single measurement for each scanned pixel. In addition, we have discussed the system components and the post-processing parameters that affect the depth profiling accuracy and the 3D reconstruction operation the most. Results showed that the instrument response function of the system and the time gate window width in a post-processing phase are playing the most important role in determining the axial (depth) accuracy. We believe that our system will enable a whole new class of Raman applications that will allow simultaneous 3D chemical geometric representation at the cm-level during Raman operations.

An intelligent alkyne-tag for Raman imaging of living cells: graphdiyne-encapsulated Au nanospheres.

A new graphdiyne-encapsulated Au nanosphere (Au@GDY) material was fabricated, which possessed an amplified Raman signal of acetylene linkage and produced bright, stable, and distinct signals in the cellular Raman-silent region. Its signal repeatability is far superior to that of alkyne-containing molecules. This work provides promise as an alkyne-tag for Raman imaging of living cells.

Quantitative imaging methods for heterogeneous multi-component films.

The drying of multi-component dispersions is a common phenomenon in a variety of everyday applications, including coatings, inks, processed foods, and cosmetics. As the solvent evaporates, the different components may spontaneously segregate laterally and/or in depth, which can significantly impact the macroscopic properties of the dried film. To obtain a quantitative understanding of these processes, high-resolution analysis of segregation patterns is crucial. Yet, current state-of-the-art methods are limited to transparent, non-deformable labeled colloids, limiting their applicability. In this study, we employ three techniques that do not require customized samples, as their imaging contrast relies on intrinsic variations in the chemical nature of the constituent species: confocal Raman microscopy, cross-sectional Raman microscopy, and a combination of scanning electron microscopy and energy dispersive X-ray analysis (SEM-EDX). For broad accessibility, we offer a thorough guide to our experimental steps and data analysis methods. We benchmark the capabilities on a film that dries homogeneously at room temperature but exhibits distinct segregation features at elevated temperature, notably self-stratification, i.e., autonomous layer formation, due to a colloidal size mismatch. Confocal Raman microscopy offers a direct means to visualize structures in three dimensions without pre-treatment, its accuracy diminishes deeper within the film, making cross-sectional Raman imaging and SEM-EDX better options. The latter is the most elaborate method, yet we show that it can reveal the most subtle and small-scale microseparation of the two components in the lateral direction. This comparative study assists researchers in choosing and applying the most suitable technique to quantify structure formation in dried multi-component films.

Lateral Flow Immunoassay Strip Based on Confocal Raman Imaging for Ultrasensitive and Rapid Detection of COVID-19 and Bacterial Biomarkers

Lateral Flow Immunoassay Strip Based on Confocal Raman Imaging for Ultrasensitive and Rapid Detection of COVID-19 and Bacterial Biomarkers

Exploration of utility of combined optical photothermal infrared and Raman imaging for investigating the chemical composition of microcalcifications in breast cancer.

Microcalcifications play an important role in cancer detection. They are evaluated by their radiological and histological characteristics but it is challenging to find a link between their morphology, their composition and the nature of a specific type of breast lesion. Whilst there are some mammographic features that are either typically benign or typically malignant often the appearances are indeterminate. Here, we explore a large range of vibrational spectroscopic and multiphoton imaging techniques in order to gain more information about the composition of the microcalcifications. For the first time, we validated the presence of carbonate ions in the microcalcifications by O-PTIR and Raman spectroscopy at the same time, the same location and the same high resolution (0.5 μm). Furthermore, the use of multiphoton imaging allowed us to create stimulated Raman histology (SRH) images which mimic histological images with all chemical information. In conclusion, we established a protocol for efficiently analysing the microcalcifications by iteratively refining the area of interest.

Silent Raman imaging of highly effective anti-bacterial activity synchronous with biofilm breakage using poly(4-cyanostyrene)@silver@polylysine nanocomposites.

Biofilms are known to be a great challenge for their anti-bacterial activity as they obstruct drug action for deeper and more thorough bacteria-killing effects. Therefore, developing highly effective antibacterial agents to destroy biofilms and eradicate bacteria is of great significance. Herein, a new type of nanocomposites (denoted as poly(4-cyanostyrene)@silver@polylysine) is proposed, in which polylysine (PLL) could rapidly capture the biofilms and exhibit excellent antibacterial efficacy together with decorated silver (Ag) nanoparticles (NPs) through the charge effect and Ag+ release. Notably, nearly 100% antibacterial rates against Gram-positive bacterium (Staphylococcus aureus, S. aureus) and Gram-negative bacterium (Escherichia coli, E. coli) were achieved. More importantly, poly(4-cyanostyrene) with biological silent Raman imaging capacity is able to illustrate the relationship between antibacterial efficiency and biofilm breakage. In short, such novel nanocomposites can improve the bioavailability of each component and display tremendous potential in antibacterial applications.

Tip-enhanced Raman imaging of plasmon-driven dimerization of 4-bromothiophenol on nickel-decorated gold nanoplate bimetallic nanostructures.

We used tip-enhanced Raman spectroscopy (TERS) to examine plasmon-driven dimerization of 4-bromothiophenol (4-BTP) into thiophenol (TP) and 4,4'-biphenyldithiol (4,4'-BPDT) on Au and Ni@AuNPs. TERS revealed that cross-coupling of these molecular reactants into 4,4'-BPDT occurred primarily on Ni nano islands rather than the surrounding Au on the surface of Ni@AuNPs.

Multiple deuteration of triphenylphosphine and live-cell Raman imaging of deuterium-incorporated Mito-Q.

All aromatic C-H bonds of triphenylphosphine (PPh3) were efficiently replaced by C-D bonds using Ru/C and Ir/C co-catalysts in 2-PrOH and D2O, an inexpensive deuterium source. Furthermore, non-radioactive and safe deuterium-incorporated Mito-Q (drug candidate) was prepared from deuterated PPh3 and used for the live-cell Raman imaging to evaluate the mitochondrial uptake.

Decorin improves human pancreatic β-cell function and regulates ECM expression in vitro

Transplantation of islets of Langerhans is a promising alternative treatment strategy in severe cases of type 1 diabetes mellitus; however, the success rate is limited by the survival rate of the cells post-transplantation. Restoration of the native pancreatic niche during transplantation potentially can help to improve cell viability and function. Here, we assessed for the first time the regulatory role of the small leucine-rich proteoglycan decorin (DCN) in insulin secretion in human β-cells, and its impact on pancreatic extracellular matrix (ECM) protein expression in vitro. In depth analyses utilizing next-generation sequencing as well as Raman microspectroscopy and Raman imaging identified pathways related to glucose metabolism to be upregulated in DCN-treated cells, including oxidative phosphorylation within the mitochondria as well as proteins and lipids of the endoplasmic reticulum. We further showed the effectiveness of DCN in a transplantation setting by treating collagen type 1-encapsulated β-cell-containing pseudo-islets with DCN. Taken together, in this study, we demonstrate the potential of DCN to improve the function of insulin-secreting β-cells while reducing the expression of ECM proteins affiliated with fibrotic capsule formation, making DCN a highly promising therapeutic agent for islet transplantation.

Laser-Induced Selective Metallization on Polymers for Both NIR and UV Lasers: Preparing 2D and 3D Circuits

Laser-induced selective metallization (LISM) technologies have received much attention because of their applications in metallized patterns and two-dimensional (2D) and three-dimensional (3D) circuits. Our work proposed a new laser sensitizer, copper pyrophosphate (Cu2P2O7·3H2O), which can perform LISM for both 1064 nm near-infrared (NIR) laser and 355 nm ultraviolet (UV) laser. Conductivities of copper layers obtained by NIR and UV LISM on the ABS/Cu2P2O7 composite were 1.52 × 107 and 1.47 × 107 Ω–1·m–1, respectively. The copper layer adhesion to composites reached the 5B Level of ASTM D3359. Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed that Cu2P2O7·3H2O changed color due to the loss of crystalline water during composite preparation. Scanning electron microscopy (SEM), optical microscopy (OM), and Raman imaging indicated that a microrough structure and amorphous carbon appeared on composites. X-ray photoelectron spectroscopy (XPS) revealed that Cu2P2O7·3H2O was reduced to Cu0 after laser activation, and this Cu0 was the active species to induce electroless copper plating (ECP). About 54.39 and 30.57% of Cu2+ were reduced to Cu0 after NIR and UV laser activation. NIR laser can generate more Cu0 due to a higher thermal effect, so the speed of ECP is faster. In addition, decorative patterns and interdigitated capacitor patterns were successfully fabricated on 3D plastic parts by LISM, demonstrating potential application prospects.

Raman imaging monitors the time-resolved response of A. thaliana to the artificial inhibition of PSII

The short-term (0–96 h) response of A. thaliana to the oxidative stress induced by PSII inhibitor metribuzin was examined using Raman spectroscopy. Whole leaves of wildtype (WT, Col-0) and ros1 mutant were scanned and changes in carotenoids were examined. Strong differences in Raman intensity distributions between WT and ros1 were observed. A stronger decrease of carotenoid v1(C=C) band intensity across the leaf was observed in ros1 after 48 h of exposure to metribuzin. It can be assumed that higher sensitivity to oxidative stress in ros1 mutant results in significantly faster degradation of carotenoids.

Raman Spectroscopy and Imaging Studies of Human Digestive Tract Cells and Tissues—Impact of Vitamin C and E Supplementation

Cancers of digestive tract such as colorectal cancer (CRC) and gastric cancer (GC) are the most commonly detected types of cancer worldwide and their origin can be associated with oxidative stress conditions. Commonly known and followed antioxidants, such as vitamin C and E, are widely considered as potential anti-cancer agents. Raman spectra have great potential in the biochemical characterization of matter based on the fact that each molecule has its own unique vibrational properties. Raman spectroscopy allows to precisely characterize components (proteins, lipids, nucleic acids). The paper presents the application of the Raman spectroscopy technique for the analysis of tissue samples and cells of the human colon and stomach. The main goal of this study is to show the differences between healthy and cancerous tissues from the human digestive tract and human normal and cancer colon and gastric cell lines. The paper presents the spectroscopic characterization of normal colon cells, CCD-18 Co, in physiological and oxidative conditions and effect of oxidative injury of normal colon cells upon supplementation with vitamin C at various concentrations based on Raman spectra. The obtained results were related to the Raman spectra recorded for human colon cancer cells-CaCo-2. In addition, the effect of the antioxidant in the form of vitamin E on gastric cancer cells, HTB-135, is presented and compared with normal gastric cells-CRL-7869. All measured gastric samples were biochemically and structurally characterized by means of Raman spectroscopy and imaging. Statistically assisted analysis has shown that normal, ROS injured and cancerous human gastrointestinal cells can be distinguished based on their unique vibrational properties. ANOVA tests, PCA (Principal Component Analysis) and PLSDA (Partial Least Squares Discriminant Analysis) have confirmed the main role of nucleic acids, proteins and lipids in differentiation of human colon and stomach normal and cancer tissues and cells. The conducted research based on Raman spectra proved that antioxidants in the form of vitamin C and E exhibit anti-cancer properties. In consequence, conducted studies proved that label-free Raman spectroscopy may play an important role in clinical diagnostic differentiation of human normal and cancerous gastrointestinal tissues and may be a source of intraoperative information supporting histopathological analysis.

Tracking spatiotemporal evolution of cementitious carbonation via Raman imaging

AbstractCarbonation of cementitious systems is an important phenomenon from both durability (rebar corrosion) and greenhouse emissions (CO2 sequestration) perspectives. Several analytical techniques are well‐established for measuring the kinetics of this dynamic phenomenon, ranging from measuring the pH change to quantifying the calcite content over time. However, the majority of these methods (with the exception of electron imaging) rely on bulk measurements which may miss the fine, microstructural changes that occur during carbonation. In this study, we investigate and evaluate the potential of Raman imaging to follow the spatiotemporal evolution of carbonation in cementitious pastes. By analysing mm‐scale maps (1 mm × 1 mm) at the micron‐scale resolution, we find that nearly ~40% of the sample surface was covered with calcite at the end of a 2‐week carbonation exposure. Simultaneously, the portlandite content declined from ~15% to 5% in the same period, suggesting portlandite consumption for the sake of calcite growth. Finally, the difference between the calcite growth rate and portlandite consumption rate strongly suggests simultaneous carbonation of CSH and ettringite—a finding that is in agreement with recent experimental investigations of carbonation kinetics. Overall, these new results on the spatiotemporal evolution of cementitious microstructure pave the way for utilizing Raman imaging for understanding dynamic phenomena in complex systems.

Growth of ultrathin Bi2Se3 films by molecular beam epitaxy

Bi 2 Se 3 is a widely studied 3D topological insulator having potential applications in optics, electronics, and spintronics. When the thickness of these films decreases to less than approximately 6 nm, the top and bottom surface states couple, resulting in the opening of a small gap at the Dirac point. In the 2D limit, Bi2Se3 may exhibit quantum spin Hall states. However, growing coalesced ultrathin Bi2Se3 films with a controllable thickness and typical triangular domain morphology in the few nanometer range is challenging. Here, we explore the growth of Bi2Se3 films having thicknesses down to 4 nm on sapphire substrates using molecular beam epitaxy that were then characterized with Hall measurements, atomic force microscopy, and Raman imaging. We find that substrate pretreatment—growing and decomposing a few layers of Bi2Se3 before the actual deposition—is critical to obtaining a completely coalesced film. In addition, higher growth rates and lower substrate temperatures led to improvement in surface roughness, in contrast to what is observed for conventional epitaxy. Overall, coalesced ultrathin Bi2Se3 films with lower surface roughness enable thickness-dependent studies across the transition from a 3D-topological insulator to one with gapped surface states in the 2D regime.

Graphene nano-sieves by femtosecond laser irradiation

The formation of nano-pores in graphene crystal structure is alternative way to engineer its electronic properties, chemical reactivity, and surface interactions, enabling applications in technological fields such as sensing, energy and separation. The past few years, nano-perforation of graphene sheets has been accomplished by a variety of different methods suffering mainly from poor scalability and cost efficiency issues. In this work, we introduce an experimental protocol to engineer nanometer scale pores in CVD graphene membranes under ambient conditions, using low power ultra-short laser pulses and overcoming the drawbacks of other perforation techniques. Using Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) we visualized and quantified the nanopore network while Raman spectroscopy is utilized to correlate the nano-perforated area with the nanotopographic imaging. We suggest that Raman imaging provides the identification of nanoporous area and, in combination with AFM, we provide solid evidence for the reproducibility of the method, since under these experimental conditions, nanopores of a certain size distribution are formed.

Frequency Changes in Terminal Alkynes Provide Strong, Sensitive, and Solvatochromic Raman Probes of Biochemical Environments.

The C≡C stretching frequencies of terminal alkynes appear in the "clear" window of vibrational spectra, so they are attractive and increasingly popular as site-specific probes in complicated biological systems like proteins, cells, and tissues. In this work, we collected infrared (IR) absorption and Raman scattering spectra of model compounds, artificial amino acids, and model proteins that contain terminal alkyne groups, and we used our results to draw conclusions about the signal strength and sensitivity to the local environment of both aliphatic and aromatic terminal alkyne C≡C stretching bands. While the IR bands of alkynyl model compounds displayed surprisingly broad solvatochromism, their absorptions were weak enough that alkynes can be ruled out as effective IR probes. The same solvatochromism was observed in model compounds' Raman spectra, and comparisons to published empirical solvent scales (including a linear regression against four meta-aggregated solvent parameters) suggested that the alkyne C≡C stretching frequency mainly reports on local electronic interactions (i.e., short-range electron donor-acceptor interactions) with solvent molecules and neighboring functional groups. The strong solvatochromism observed here for alkyne stretching bands introduces an important consideration for Raman imaging studies based on these signals. Raman signals for alkynes (especially those that are π-conjugated) can be exceptionally strong and should permit alkynyl Raman signals to function as probes at very low concentrations, as compared to other widely used vibrational probe groups like azides and nitriles. We incorporated homopropargyl glycine into a transmembrane helical peptide via peptide synthesis, and we installed p-ethynylphenylalanine into the interior of the Escherichia coli fatty acid acyl carrier protein using a genetic code expansion technique. The Raman spectra from each of these test systems indicate that alkynyl C≡C bands can act as effective and unique probes of their local biomolecular environments. We provide guidance for the best possible future uses of alkynes as solvatochromic Raman probes, and while empirical explanations of the alkyne solvatochromism are offered, open questions about its physical basis are enunciated.