Raman Imaging Research Articles

The role of cardiolipin and cytochrome c in mitochondrial metabolism of cancer cells determined by Raman imaging: in vitro study on the brain glioblastoma U-87 MG cell line.

In this paper, we present Raman imaging as a non-invasive approach for studying changes in mitochondrial metabolism caused by cardiolipin-cytochrome c interactions. We investigated the effect of mitochondrial dysregulation on cardiolipin (CL) and cytochrome c (Cyt c) interactions for a brain cancer cell line (U-87 MG). Mitochondrial metabolism was monitored by checking the intensities of the Raman bands at 750 cm-1, 1126 cm-1, 1310 cm-1, 1337 cm-1, 1444 cm-1 and 1584 cm-1. The presented results indicate that under pathological conditions, the content and redox status of Cyt c in mitochondria can be used as a Raman marker to characterize changes in cellular metabolism. This work provides evidence that cardiolipin-cytochrome c interactions are crucial for mitochondrial energy homeostasis by controlling the redox status of Cyt c in the electron transport chain, switching from disabling Cyt c reduction and enabling peroxidase activity. This paper provides experimental support for the hypothesis of how cardiolipin-cytochrome c interactions regulate electron transfer in the respiratory chain, apoptosis and mROS production in mitochondria.

Tracking trash to treasure: in situ monitoring of single microbial cell oil biosynthesis from waste cooking oil using Raman spectroscopy and imaging.

Waste cooking oil is a major pollutant that contaminates terrestrial and aquatic bodies which is generated from household kitchens and eateries. The bioremediation of waste cooking oil (WCO) into microbial oil, also known as single microbial cell oil (SMCO), can be accomplished by oleaginous microbes. Conventional methods excel in SMCO analysis but lack efficacy for in situ or lysis-free monitoring of nascent SMCO synthesis and turnover. To bridge this knowledge gap, this study shows the applicability of Raman reverse stable isotope probing (RrSIP) in monitoring time-dependent nascent SMCO synthesis and assimilation in Yarrowia lipolytica, an oleaginous yeast grown in hydrophilic (glucose) as well as hydrophobic carbon sources (cooking oil and waste cooking oil). This study also combines the RrSIP approach with Raman imaging for temporal visualization of the distribution and turnover dynamics of the SMCO pool in a single cell. Our finding provides a unique perspective utilizing optical spectroscopy methods for lysis-free SMCO analysis and holds potential for prospective utility as an adjunct tool in bioprocess and biofuel industries.

Visualization of Crystallinity and Intermolecular Interactions of Polymers Using Raman Spectroscopy and Raman Imaging

Visualization of Crystallinity and Intermolecular Interactions of Polymers Using Raman Spectroscopy and Raman Imaging

Alkyne-tagged imidazolium-based membrane cholesterol analogs for Raman imaging applications.

Cholesterol is an important lipid playing a crucial role in mediating essential cellular processes as well as maintaining the basic structural integrity of biological membranes. Given its vast biological importance, there is an unabated need for sophisticated strategies to investigate cholesterol-mediated biological processes. Raman-tagged sterol analogs offer the advantage of being visualizable without the need for a bulky dye that potentially affects natural membrane integration and cellular interactions as it is the case for many conventionally used fluorescent analogs. Herein, we report a series of alkyne-tagged imidazolium-based cholesterol analogs (CHIMs) with large Raman scattering cross-sections that readily integrate into HEK cells and primary monocyte-derived macrophages and allow (multiplexed) cellular Raman imaging. We envision Raman-tagged CHIM analogs to be a powerful platform for the investigation of cholesterol-mediated cellular processes complementary to other established methods, such as the use of fluorescent analogs.

Raman and Polarization-sensitive digital holographic imaging for rapid and label-free prostate cancer diagnosis

In this study, we report the results of two non-invasive optical methods, Raman microscopy (RM) and polarization-sensitive digital holographic imaging (PSDHI), for distinguishing prostate cancer cells from healthy ones. RM reveals cancer cells metabolize glucose faster, storing it as fatty acids and cholesteryl esters in lipid droplets (LDs). On the other hand, PSDHI shows significant morphological changes in LDs in glucose-incubated cancer cells, including number, volume, and refractive index. High birefringence in cancer LDs under perpendicular polarizations was observed, enabling fast discrimination with over 90% accuracy. PSDHI results align closely with Raman microscopy, suggesting its potential as a promising, high-speed technique for cancer screening purposes.

Morphology of impact polypropylene copolymer extruded cast film revealed by confocal Raman imaging.

An impact polypropylene copolymer (IPC), composed of polypropylene (PP) and ethylene-propylene copolymer (EPC), was synthesized through two-stage in-reactor polymerization. A systematic investigation of the crystalline structure, thermal behavior, morphology, and tensile properties of the IPC extruded cast film was conducted. Specifically, the morphology of EPC was obtained by confocal Raman imaging by depicting the spatial distribution of the Raman band located at 1064 cm-1. The EPC phase exhibits fibrous morphology with the long axis aligning along the machine direction (MD). A three-dimensional (3D) heterogeneous structure of the IPC cast film obtained by confocal Raman imaging confirms that the fibrous EPC phase is dispersed in a 3D framework of the PP matrix. The mesomorphic phase in the as-prepared cast film transforms to a stable α-form crystal after annealing at 130 °C, which improves the yield strength but decreases the elongation of the cast film. The WAXD and SAXS results indicate that there is no obvious orientation of the crystallites. Thus, the anisotropy of tensile properties in the MD and transverse directions is closely related to the anisotropic phase morphology at the micrometer scale. The results reveal that the mechanical performances of IPC films are determined by the crystalline structure of the PP matrix and the morphology.

High-speed 1-D and 2-D Raman scattering measurement for quantitative characterization of transient hydrogen jets

This paper reports the first quantitative characterization of transient hydrogen (H2) jets issued from a single-hole injector into nitrogen (N2), using high-speed 1-D and 2-D Raman scattering techniques. By employing the pulse-burst laser as the light source together with the low-noise EMCCD cameras running in subframe burst gating mode for signal collection, single-shot 1-D mole fraction of H2 (xH2) deduced from the ratio of H2 and N2 Raman scattering intensities, is obtained at repetition rate of 50 kHz. By changing the injection conditions, transient jet behaviors of both subsonic and highly under-expanded jets are well captured in single shots of 1-D, and the dynamic of the oblique shocks is visualized by local peaks in relative H2 number density. Statistical information regarding the jet behaviors is also obtained by repeating the measurement at same injection conditions, and the mixing between H2 and N2 can be directly evaluated by the average xH2. A coefficient of variance (COV) of 1.6 to 6 % is measured for all the conditions near the core region of the jets at late injection time, which demonstrates a good repeatability of the 1-D line measurements during the quasi-steady injection period. Using high-speed CMOS cameras, 2-D Raman imaging of transient H2 jets is performed at repetition rate of 10 kHz. Results in single shots demonstrate the vortices formation at the start of injection leading to turbulent mixing layers at later stages. Due to the low SNR, the accuracy and precision of the 2-D measurements is limited, and a one-to-one comparison of statistical results shows a maximum deviation of approximately 15 % from the more accurate 1-D measurements.

In situ single-droplet analysis of emulsified fat using confocal Raman microscopy: insights into crystal network formation within spatial resolution.

Fat crystallization is one of the predominant factors influencing the structure and properties of fat-containing emulsions. In the present study, the role of emulsifiers on fat crystallization dynamics within droplet multiphase systems was evaluated via single-droplet analysis, taking advantage of the non-destructive properties of confocal Raman microscopy. Palm oil droplets dispersed in water were used as a model system, due to palm oil's well-known crystallization properties. Emulsion droplets of the same size were generated using two different emulsifiers (Whey Protein Isolate and Tween 60), at various concentrations. Fast and slow cooling treatments were applied to affect fat crystallisation and network formation as well as droplet morphology, and crystallization dynamics. Raman imaging analysis demonstrated that the chemical structure and concentration of the emulsifier significantly influenced both crystal nucleation within the droplets, as well as the spatial distribution and morphology of the fat crystal network. Additionally, analysis of the spectra of the crystallized phase provided essential information regarding the impact of the emulsifiers on the microstructure, degree of structural order, and structural arrangements of the fat crystal networks. Furthermore, by performing single droplet analysis during cooling it was possible to observe shape distortions in Tween 60 stabilized droplets, as a consequence of the formation of a three-dimensional network of fat crystals that strongly interacted with the interface. On the other hand, the droplets retained their shape when whey proteins were absorbed at the interface. Confocal Raman microscopy, in combination with polarized light microscopy, is, therefore, a well-suited tool for in situ, single-droplet analysis of emulsified oil systems, providing essential information about emulsified fat crystallization dynamics, contributing to better understanding and designing products with enhanced structure and function.

Viewing 3D spatial biology with highly-multiplexed Raman imaging: from spectroscopy to biotechnology.

Understansding complex biological systems requires the simultaneous characterization of a large number of interacting components in their native 3D environment with high spatial resolution. Highly-multiplexed Raman imaging is an emerging general strategy for detecting biomarkers with scalable multiplexity and ultra-sensitivity based on a series of stimulated Raman scattering (SRS) techniques. Here we review recent advances in highly-multiplexed Raman imaging and how they contribute to the technological revolution in 3D spatial biology, focusing on the developmental pathway from spectroscopy study to biotechnology invention. We envision highly-multiplexed Raman imaging is taking off, which will greatly facilitate our understanding in biological and medical research fields.

Surface-enhanced Raman scattering detection of thiram and ciprofloxacin using chitosan-silver coated paper substrates.

Fast detection of contaminants of emerging concern (CECs) in water resources is of great environmental interest. Ideally, sustainable materials should be used in water quality monitoring technologies implemented for such purposes. In this regard, the application of bio-based materials aimed at the fabrication of analytical platforms has become of great importance. This research merges both endeavors by exploring the application of chitosan-coated paper, decorated with silver nanoparticles (AgNPs), on surface-enhanced Raman scattering (SERS) spectroscopy studies of two distinct types of CECs dissolved in aqueous samples: an antibiotic (ciprofloxacin) and a pesticide (thiram). Our results indicate the superior SERS performance of biocoated substrates compared to their non-coated paper counterparts. The detection limits achieved for thiram and ciprofloxacin using the biocoated substrates were 0.024 ppm and 7.7 ppm, respectively. The efficient detection of both analytes is interpreted in terms of the role of the biopolymer in promoting AgNPs assemblies that result in local regions of enhanced SERS activity. Taking advantage of these observations, we use confocal Raman microscopy to obtain Raman images of the substrates using ciprofloxacin and thiram as molecular probes. We also demonstrate that these biobased substrates can be promising for on-site analysis when used in conjunction with portable Raman instruments.

In situ monitoring of the shikimate pathway: a combinatorial approach of Raman reverse stable isotope probing and hyperspectral imaging.

Sensing and visualization of metabolites and metabolic pathways in situ are significant requirements for tracking their spatiotemporal dynamics in a non-destructive manner. The shikimate pathway is an important cellular mechanism that leads to the de novo synthesis of many compounds containing aromatic rings of high importance such as phenylalanine, tyrosine, and tryptophan. In this work, we present a cost-effective and extraction-free method based on the principles of stable isotope-coupled Raman spectroscopy and hyperspectral Raman imaging to monitor and visualize the activity of the shikimate pathway. We also demonstrated the applicability of this approach for nascent aromatic amino acid localization and tracking turnover dynamics in both prokaryotic and eukaryotic model systems. This method can emerge as a promising tool for both qualitative and semi-quantitative in situ metabolomics, contributing to a better understanding of aromatic ring-containing metabolite dynamics across various organisms.

Imaging specific proteins in living cells with small unnatural amino acid attached Raman reporters.

Fluorescence labeling via fluorescent proteins (FPs) or immunofluorescence has been routinely applied for microscopic imaging of specific proteins. However, due to these over-weight and oversized labels (e.g. GFP, 238 aa, 27 kDa, ∼4 nm in size), the potential physiological malfunctions of the target proteins are largely underestimated in living cells. Herein, for living cells, we report a small and minimally-invasive Raman reporter (about 2 aa and <1 kDa), which can be site-specifically introduced into proteins by genetic codon expansion. After a single unnatural amino acid (UAA) is precisely incorporated into the target protein, the strained alkyne can rapidly undergo copper-free Diels-Alder cycloaddition reactions with the tetrazine-functionalized Raman reporter, which features a fine vibrational spectrum in contrast to fluorescence. In our experimental results, the UAA-based Raman tag was successfully incorporated into vimentin, histone 3.3 and huntingtin (Htt74Q) proteins in living HeLa cells and further utilized for stimulated Raman imaging. The site-specific bioorthogonal fusion of small Raman tags with intracellular proteins will pave the way for minimally-invasive protein labeling and multi-color imaging in living cells.

Advancing triage of acute lymphoblastic leukaemia subtypes diagnosis: label-free Raman spectroscopy for precise single-cell phenotyping and subtype classification.

Acute lymphoblastic leukaemia (ALL) is a complex disease in pediatric oncology, necessitating accurate diagnostic strategies for effective treatment planning. The ability to differentiate between B-cell ALL (B-ALL) and T-cell ALL (T-ALL) is crucial for targeted interventions. However, current diagnostic methods are time-consuming and require rapid, dependable tests. This study explores the potential of label-free Raman imaging coupled with chemometrics for rapid blast phenotyping of B-ALL and T-ALL. Our findings demonstrate the efficacy of Raman spectroscopy in sensitively and specifically screening and classifying ALL, as well as its rapidity and reliability. The obtained molecular information allows for label-free and precise leukaemia diagnosis at the single-cell level, surpassing the capabilities of traditional diagnostic techniques. Raman spectra of cancer cells reveal distinctive molecular signatures, specifically heightened protein and nucleic acid content, revealing molecular signatures unique to leukemic phenotypes. Based on that, they could be distinguished from each other and their normal B and T lymphocyte counterparts. This research underscores the analytical power of Raman spectroscopy, positioning it as a valuable tool for identifying and classifying pediatric ALL subtypes. The potential translational applications in clinical practice offer a promising avenue for an expedited and accurate leukaemia diagnosis, paving the way for more targeted and personalised therapeutic approaches.

In-line Raman imaging of mixing by herringbone grooves in microfluidic channels.

The control over fluid flow achievable in microfluidic devices creates opportunities for applications in many fields. In simple microchannels, flow is purely laminar when one solvent is used, and hence, achieving reliable mixing is an important design consideration. Integration of structures, such as grooves, into the channels to act as static mixers is a commonly used approach. The mixing induced by these structures can be validated by determining concentration profiles in microfluidic channels following convergence of solvent streams from separate inlets. Spatially resolved characterisation is therefore necessary and requires in-line analysis methods. Here we report a line-focused illumination approach to provide operando, spatially resolved Raman spectra across the width of channels in the analysis of single- and multi-phase liquid systems and chemical reactions. A scientific complementary metal oxide semiconductor (sCMOS) sensor is used to overcome smearing encountered during spectral readout of images with CCD sensors. Isotopically labelled probes, in otherwise identical flow streams, show that z-confocality limits the spatial resolution and certainty as to the extent of mixing that can be achieved. These limitations are overcome using fast chemical reactions between reagents entering a microchannel in separate solvent streams. We show here that the progression of a chemical reaction, for which only the product is observable, is a powerful approach to determine the extent of mixing in a microchannel. Specifically resonance enhancement of Raman scattering from a product formed allows for determination of the true efficiency of mixing over the length and width of microchannels. Raman spectral images obtained by line-focused illumination show onset of mixing by observing the product of reagents entering from the separate inlets. Mixing is initially off-centre and immediately before the apex of the first groove of the static mixer, and then evolves along the entire width of the channel after a full cycle of grooves.

New insights into lipid and fatty acid metabolism from Raman spectroscopy.

One of the challenges facing biology is to understand metabolic events at a single cellular level. While approaches to examine dynamics of protein distribution or report on spatiotemporal location of signalling molecules are well-established, tools for the dissection of metabolism in single living cells are less common. Advances in Raman spectroscopy, such as stimulated Raman scattering (SRS), are beginning to offer new insights into metabolic events in a range of experimental systems, including model organisms and clinical samples, and across a range of disciplines. Despite the power of Raman imaging, it remains a relatively under-used technique to approach biological problems, in part because of the specialised nature of the analysis. To raise the profile of this method, here we consider some key studies which illustrate how Raman spectroscopy has revealed new insights into fatty acid and lipid metabolism across a range of cellular systems. The powerful and non-invasive nature of this approach offers a new suite of tools for biomolecular scientists to address how metabolic events within cells informs on or underpins biological function. We illustrate potential biological applications, discuss some recent advances, and offer a direction of travel for metabolic research in this area.

Differentiation of Salivary Gland and Salivary Gland Tumor Tissue via Raman Imaging Combined with Multivariate Data Analysis.

Salivary gland tumors (SGTs) are a relevant, highly diverse subgroup of head and neck tumors whose entity determination can be difficult. Confocal Raman imaging in combination with multivariate data analysis may possibly support their correct classification. For the analysis of the translational potential of Raman imaging in SGT determination, a multi-stage evaluation process is necessary. By measuring a sample set of Warthin tumor, pleomorphic adenoma and non-tumor salivary gland tissue, Raman data were obtained and a thorough Raman band analysis was performed. This evaluation revealed highly overlapping Raman patterns with only minor spectral differences. Consequently, a principal component analysis (PCA) was calculated and further combined with a discriminant analysis (DA) to enable the best possible distinction. The PCA-DA model was characterized by accuracy, sensitivity, selectivity and precision values above 90% and validated by predicting model-unknown Raman spectra, of which 93% were classified correctly. Thus, we state our PCA-DA to be suitable for parotid tumor and non-salivary salivary gland tissue discrimination and prediction. For evaluation of the translational potential, further validation steps are necessary.

The role of glucose and fructose on lipid droplet metabolism in human normal bronchial and cancer lung cells by Raman spectroscopy

Fructose is one of the most important monosaccharides in the human diet that the human body needs for proper metabolism. This paper presents an approach to study biochemical changes caused by sugars in human normal bronchial cells (BEpiC) and human cancer lung cells (A549) by Raman spectroscopy and Raman imaging. Results after supplementation of human bronchial and lung cells with fructose are also discussed and compared with results obtained for pure human bronchial and lung cells. Based on Raman techniques we have proved that peaks at 750 cm−1, 1126 cm−1, 1444 cm−1, 1584 cm−1 and 2845 cm−1 can be treated as biomarkers to monitor fructose changes in cells. Results for fructose have been compared with results for glucose. Raman analysis of the bands at 750 cm−1, 1126 cm−1, 1584 cm−1 and 2845 cm−1 for pure BEpiC and A549 cells and BEpiC and A549 after supplementation with fructose and glucose are higher after supplementation with fructose in comparison to glucose. The obtained results shed light on the uninvestigated influence of glucose and fructose on lipid droplet metabolism by Raman spectroscopy methods.

Gold-silica plasmonic nanobones with tunable size and optical bimodality for bioimaging

Silica-coated gold nanotags were developed for multimodal Raman and fluorescent imaging. These nanotags, shaped like bones, offer significant absorption at 700 nm, aligning with the first window of tissue transparency. Gold nanobones were modified with cyanine 5.5 and 7 dyes and a silica shell for stability and biocompatibility via a modified Stober method involving tetraethoxysilane hydrolysis. Folic acid was attached on outer surface of nanotags to serve as a model delivery vector. The nanotags were characterized by dynamic light scattering, transmission electron microscopy, and spectroscopy. These methods showed that a thickness of the silica shell can be precisely controlled via a concentration of tetraethoxysilane. Surface-enhanced Raman scattering and emission spectra were registered and demonstrated the intensity dependent on the distance between the dye and core. Raman mapping of cell samples highlighted the nanotags bioimaging potential and revealed the partial nanotags agglomeration post-cell uptake accompanied by formation of optical hot-spots. Cell viability assays with HeLa and HEP 2 G cell lines suggested the nanotags safety below 0.2 mg/L concentration. These findings highlight the benefits of silica-coated gold nanotags and open the doors in advanced medical imaging.

Investigation of interfacial mass transfer phenomena applying non-invasive Raman imaging and density gradient theory

This study presents a novel non-invasive Raman measurement setup for investigating diffusion and mass transfer processes at liquid/liquid interfaces, based on a photometric setup utilizing customized photon multiplier (CPMs). The results highlight the effectiveness, reliability and applicability of this setup in visualizing and analyzing acetone mass transfer across the aqueous-organic interface. The study focuses on the liquid/liquid extraction of acetone from the aqueous phase into the organic phase, using toluene as the organic solvent. The study's key findings indicate that – in the absence of forced convection – mass transfer across the interface is not the limiting factor in the extraction process; instead, diffusion in the bulk phase takes precedence. Acetone's rapid local transport from the aqueous to the organic phase is observed, with a temporary concentration minimum on the aqueous side of the interface. In the organic phase, local equilibrium is reached near the interface first, only after which acetone disperses into the bulk phase. This dual mechanism—fast interfacial mass transfer and slower bulk-phase diffusion—marks a significant feature of the considered extraction process, which were further substantiated by dynamic concentration gradient theory simulations. The research further demonstrates the feasibility of upscaling interfacial mass transfer simulations using adaptive meshing. However, macroscopic effects like induced convection challenge purely diffusive models. The simulations confirm rapid enrichment of acetone at the interface, followed by slower mass transfer through the interface and bulk phase diffusion, consistent with the experimental data. Experimental and modeled results align well with literature data for liquid/liquid extraction, thereby validating the accuracy of the presented novel diffusion measurement setup and demonstrating its potential to investigate mass transfer phenomena near liquid/liquid interfaces.

Comparative Characterization of Reference Market Product and Generic Product Developed using Reverse Engineering Methodology

The present study is a comparative analysis between the reference market product, Xarelto® 20 mg film-coated tablets, and a generic product developed using reverse engineering methodology, employing low-shear ethanolic granulation. The primary objective in formulating and optimizing this generic product was to attain a formulation that exhibits both physical and chemical stability, closely resembling the innovator or market reference drug product. Both reference and generic products were evaluated using FTIR, TGA, DSC, XRD, SEM, EDX/EDS, Raman mapping, Raman imaging and contact angle measurement studies and were compared. Similar characteristic peaks were observed in the IR spectra of the generic product (RX-LS [AH]) to that of the reference product. Similar crystallinity was detected between reference and generic products. The TG-DSC analysis confirmed the presence of a similar endothermic peak of the generic product to that of the reference product. SEM images showed the presence of deformed granules in both, generic and reference products due to the compression force during tablet production. The specific and major elements present in both samples were C, N, O, Na, Mg, S and Cl. The API and excipient distribution within the reference product and the generic product is closely similar confirmed through the Raman study. Test (θ = 17.3) and reference products (θ = 17.9) showed contact angles around 17 degrees, it indicates that both products have similar wettability capacity, in other words, similar solubility and dissolution behavior. The current study demonstrated the excellent similarity of generic Rivaroxaban 20 mg film-coated tablets with reference products. The exactly similar characteristics of generic products will definitely boost confidence of successful bioequivalence studies and reduce developmental cost and time as there is no a chance of product failure which will lead to redevelopment.