Raman Imaging Research Articles

Using clustering as pre-processing in the framework of signal unmixing for exhaustive exploration of archaeological artefacts in Raman imaging

Analytical chemistry on archaeological material is an essential part of modern archaeological investigations and from year to year, instrumental improvement has made it possible to generate data at a high spatial and temporal frequency. In particular, Raman spectral imaging can be successfully applied in archaeological research by its simplicity of implementation to study past human societies through the analysis of their material remains. This technique makes it possible to simultaneously obtain spatial and spectral information by preserving sample integrity. However, because of the inherent complexity of the samples in Archaeology (e.g. seniority, fragility, lack or full absence of any information about its composition), chemical interpretation can be difficult at first glance. Indeed, specific problems of spectral selectivity related to unexpected chemical compounds could appear due to their state of conservation. Furthermore, detecting minor compounds becomes challenging as major components impose their contributions in the acquired spectra. Therefore, a relevant chemometric approach has been introduced in this context to characterize distinct spectral sources in a Raman imaging dataset of an archaeological specimen – a mosaic fragment. The fragment was unearthed during the Ruscino archaeological dig on the outskirts of Perpignan, France. It dates back to the oppidum period. The aim is to extract selective spectral information from pixel clustering analysis in order to enhance the initial optimisation step within the Multivariate Curve Resolution and Alternating Least-Squares (MCR-ALS) algorithm, a well-known signal unmixing technique. The underlying principle of the MCR-ALS is that the acquired spectra can be expressed as linear combinations of pure spectra of all individual components present in the chemical system under study. Sometimes it can be difficult to obtain the desired results through the algorithm, particularly if initial estimates of spectral or concentration profiles are inaccurate due to complex signals, noise or lack of selectivity, resulting in rank deficiency (i.e. a poor estimation of the total number of pure signals). For this reason, an innovative threshold-based clustering algorithm, combined with multiple Orthogonal Projection Approaches (OPA), has been developed to improve matrix rank investigation and thus the initialisation step of the MCR-ALS approach before optimisation. The effective analysis of Raman imaging data for an archaeological mosaic played a crucial role in uncovering significant chemical information about a particular biogenic material. This insight sheds light on the origins of mortar manufacture during the oppidum period.

Abstract 201: Morphological changes to the vulvar squamous carcinoma cell line, A431, resulting from clobetasol-induced upregulation of vimentin

Abstract Vimentin is recognized as an important marker in the epithelial-mesenchymal transition (EMT) of several cancer cell types. The overexpression of vimentin appears to coincide with increased tumor growth, migration, and poor prognosis. The vulvar cancer cell line, A431, is of squamous cell origin and displays hallmarks of a squamous cell including expression of cytokeratins 8 and 18 as well as E- and P-cadherin. We have found that treatment of A431 cells with the glucocorticoid, clobetasol, results in upregulation of vimentin which is accompanied by change to a more mesenchymal-like morphology. Despite this change in morphology, the cells continue to express cytokeratins 8 and 18. This suggests that the vimentin has a greater impact on cell morphology than does cytokeratins 8 and 18. However, we previously reported that the analogous glucocorticoid, dexamethasone, also downregulates E- and P-cadherin in these cells which may account for the observed change in morphology. Here we examine more closely the impact of vimentin on cellular morphology in the clobetasol treated A431 cells by both immunofluorescence microscopy and Raman imaging. Citation Format: Jani E. Lewis, Kia Haering, Kazushige Yokoyama. Morphological changes to the vulvar squamous carcinoma cell line, A431, resulting from clobetasol-induced upregulation of vimentin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 201.

Abstract 4168: Pre-clinical demonstration of Raman spectroscopy-assisted photothermal therapy for prostate cancer

Abstract Prostate cancer (PCa) ranks among the most prevalent malignancies in men. The combination of ultrasound and fine-needle aspiration biopsy has greatly facilitated early diagnosis and expanded the range of treatment options. Traditional treatments primarily involve anti-androgens and androgen deprivation therapy; however, a recurrence occurs in approximately 30% of cases following these interventions. Our current focus is on the exploration of photothermal therapies using nanoparticles for precise tumor ablation. Among these nanoparticles, multiwalled carbon nanotubes (MWCNTs) exhibit unique structural, optical, and thermal properties that make them exceptionally appealing for biomedical applications, especially as a foundation for photothermal therapy. Our platform employs the copper (I)-catalyzed azide alkyne cycloaddition of prostate-specific membrane antigen (PSMA)-targeting peptidomimetic (Glu-urea-Lys) ligand to the MWCNTs. We used confocal microscopy to observe a significantly enhanced binding of the targeted Glu-urea-Lys-MWCNTs to the surfaces of PSMA-expressing LNCaP cells compared to IgG-MWCNTs and to PSMA-null PC3 cells. Raman microscopy further confirmed the increased presence of Glu-urea-Lys-MWCNTs on LNCaP spheroids compared to non-targeting MWCNTs. We also confirmed enhanced photothermal cell ablation in vitro using the targeted MWCNTs. Based on in vitro evidence, we established LNCaP and PC3 xenografts in nu/nu mice and evaluated the therapeutic efficacy. Using fluorescence-based imaging, we observed the enhanced localization of Glu-urea-Lys-MWCNTs within the tumors. Furthermore, we collected the tumors and conducted ex vivo analysis using Raman microscopy for a label-free detection of the targeted MWCNTs, which complemented the fluorescence imaging and laying the foundation for real-time Raman imaging in vivo. Subsequently, we applied photothermal ablation of the tumor xenografts based on a pre-optimized treatment protocol established from COMSOL simulations and prior experiments. This treatment led to complete tumor ablation with minimal recurrence and minimal non-specific damage. In conclusion, we have assessed the effectiveness of our targeted nano-photothermal platform in a pre-clinical model of PCa. Further studies will aid in the transition of photothermal therapy to clinical trials. Moreover, we aim to investigate the role of photothermal therapy in the PCa tumor microenvironment and whether such an approach can trigger post-therapeutic activation of immune responses against the tumor. Ultimately, we anticipate that our nanotherapeutic platform will contribute to a focused therapeutic approach where targeted MWCNT-laser treatment exploits overexpressed tumor markers in conjunction with various irradiation strategies to enhance ablation while minimizing damage to surrounding tissue. Citation Format: Seung Soo Lee, Gabriel Beaudoin, Jun Hui Yeoh, Frederic Leblond, Mark Trifiro, Miltiadis Paliouras. Pre-clinical demonstration of Raman spectroscopy-assisted photothermal therapy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4168.

A Systematic Approach toward Enabling Maximal Targeting Efficiency of Cell Surface Proteins with Actively Targeted SERS Nanoparticles.

With their intricate design, nanoparticles (NPs) have become indispensable tools in the quest for precise cellular targeting. Among various NPs, gold NPs stand out with unique features such as chemical stability, biocompatibility, adjustable shape, and size-dependent optical properties, making them particularly promising for molecular detection by leveraging the surface-enhanced Raman scattering (SERS) effect. Their multiplexing abilities for the simultaneous identification of multiple biomarkers are important in the rapidly evolving landscape of diverse cellular phenotypes and biomolecular profiling. However, the challenge is ensuring that SERS NPs can effectively target specific cells and biomarkers among intricate cell types and biomolecules with high specificity. In this study, we improve the functionalization of SERS NPs, optimizing their targeting efficiency in cellular applications for ca. 160 nm NP-based probes. Spherical SERS NPs, conjugated with antibodies targeting epidermal growth factor receptor and human epidermal growth factor receptor 2, were incubated with cells overexpressing these proteins, and their specific binding potential was quantified at each stage by using flow cytometry to achieve optimal targeting efficiency. We determined that maintaining an average of 3.5 × 105 thiols per NP, 300 antibodies per NP, 18,000 NPs per cell, conducting a 15 min staining incubation at 4 °C in a shaker, and using SM(PEG)12 as a cross-linker for the NP conjugation were crucial to achieve the highest targeting efficiency. Fluorescence and Raman imaging were used with these parameters to observe the maximum ability of these NPs to efficiently target suspended cells. These highly sensitive contrast agents demonstrate their pivotal role in effective active targeting, making them invaluable for multiplexing applications across diverse biological environments.

Capturing interference variations of atomic motion in vibrational modes by non-resonant Raman images

Non-resonant Raman imaging associated with vibrational modes provides an optical means to structure and function characterisation for a single molecule, where the interference between atomic motions in a vibrational mode plays an important role. Taking the [18]annulene as a proof-of-the-concept molecule, we present here a comprehensive study on the dependence of non-resonant Raman images on interference variations involving the phase and amplitude of atomic displacements in vibrational modes. Calculations demonstrate that constructive and destructive phase interferences contribute to the characteristic merging and repulsion of Raman patterns, respectively, which should be attributed to the same/opposite signs of Raman polarisability. In addition, the imaging characteristic by constructive and destructive interferences become more significant with the increase of plasmonic size. Moreover, Raman images are highly sensitive to the subtle variations of interference induced by molecular symmetry and element substitution, highlighting the importance of amplitude variations of interference in modes for Raman images to monitor molecular structure changes. Our findings propose the capability of Raman images toward capturing interference variations in modes, serving as good references for Raman imaging to characterise various molecules and their corresponding structure changes by analysing interference effects in vibrational modes.

High-speed and wide-field 3D Raman imaging using flower-like gap-enhanced Raman tags

Raman imaging technology plays a pivotal role in biomedical research, with the pursuit of higher resolution, faster imaging speed, and a larger field of view consistently driving the development of Raman imaging systems. In this work, we propose a high-speed and wide-field three-dimensional (3D) Raman imaging method based on light field microscopy and flower-like gap-enhanced Raman tags (F-GERTs). F-GERTs with enhanced Raman signals and high stability are used to label microbial and cell samples, avoiding the problem of photobleaching. The micro-lens array and scientific camera are incorporated to capture light field Raman images containing spatial information of the sample in a single snapshot acquisition process. The 3D reconstruction algorithm can complete the processing of the light field image within 1 min, reconstructing the 3D Raman image of the sample and the precisely slice structure at different depths. This high-speed and wide-field Raman imaging technology has great potential in the field of real-time 3D Raman imaging of microorganisms and cells.

Guidelines for Raman spectroscopy and imaging techniques in food safety analysis

AbstractEnsuring the highest standards of food quality and safety is crucial for maintaining human health. This requires the creation of sophisticated and highly sensitive techniques for detecting and identifying food pollutants. Raman spectroscopy, known for its rapidity, simplicity, sensitivity, and noninvasive characteristics, has become a prominent method in the field of food safety and quality analysis. This review discusses in detail the fundamental principles, instruments, methodology, and data processing techniques of Raman spectroscopy. It specifically emphasizes the practical applications of Raman spectroscopy in assessing foodborne pathogens, toxic gases in spoiled foods, and monitoring the translocation of pesticide in plants. Finally, we discussed the development trend and challenges of Raman spectroscopy in the field of food safety analysis, aiming to provide an accessible and practical guide. This review article equips readers with essential insights into conducting and understanding Raman spectroscopy experiments, thereby contributing to development in the field of food safety analysis.

Multifaceted Characterization for the Hepatic Clearance of Graphene Oxide and Size-Related Hepatic Toxicity.

Understanding the final fate of nanomaterials (NMs) in the liver is crucial for their safer application. As a representative two-dimensional (2D) soft nanomaterial, graphene oxide (GO) has shown to have high potential for applications in the biomedical field, including in biosensing, drug delivery, tissue engineering, therapeutics, etc. GO has been shown to accumulate in the liver after entering the body, and thus, understanding the GO-liver interaction will facilitate the development of safer bio-applications. In this study, the hepatic clearance of two types of PEGylated GOs with different lateral sizes (s-GOs: ~70 nm and l-GOs: ~300 nm) was carefully investigated. We found that GO sheets across the hepatic sinusoidal endothelium, which then may be taken up by the hepatocytes via the Disse space. The hepatocytes may degrade GO into dot-like particles, which may be excreted via the hepatobiliary route. In combination with ICP-MS, LA-ICP-MS, and synchrotron radiation FTIR techniques, we found that more s-GO sheets in the liver were prone to be cleared via hepatobiliary excretion than l-GO sheets. A Raman imaging analysis of ID/IG ratios further indicated that both s-GO and l-GO generated more defects in the liver. The liver microsomes may contribute to GO biotransformation into O-containing functional groups, which plays an important role in GO degradation and excretion. In particular, more small-sized GO sheets in the liver were more likely to be cleared via hepatobiliary excretion than l-GO sheets, and a greater clearance of s-GO will mitigate their hepatotoxicity. These results provide a better understanding of the hepatic clearance of soft NMs, which is important in the safer-by-design of GO.

Label-free Raman imaging for screening of anti-inflammatory function food

Natural bioactive compounds and plant constituents are considered to have a positive anti-inflammatory effect. This study aimed to establish a screening technique for anti-inflammatory function in foods based on label-free Raman imaging. A visible anti-inflammatory analysis method based on coherent anti-Stokes Raman scattering (CARS) was established with an LPS-induced RAW264.7 cell model. Dynamic changes in proteins and lipids were determined at laser pump light wavelengths of 2956 cm−1 and 2856 cm−1, respectively. The method was applied to a plant-based formula (JC) with anti-inflammatory activity. Q-TOF-MS and HPLC analyses revealed the main active constituents of JC as quercetin, kaempferol, l-glutamine, and sodium copper chlorophyllin. In in vitro and in vivo verification experiments, JC showed significant anti-inflammatory activity by regulating the TLR4/NF-κB pathway. In conclusion, this study successfully established a label-free and visible method for screening anti-inflammatory constituents in plant-based food products, which will facilitate the evaluation of functional foods.

On-the-fly Raman microscopy guaranteeing the accuracy of discrimination

Accelerating the measurement for discrimination of samples, such as classification of cell phenotype, is crucial when faced with significant time and cost constraints. Spontaneous Raman microscopy offers label-free, rich chemical information but suffers from long acquisition time due to extremely small scattering cross-sections. One possible approach to accelerate the measurement is by measuring necessary parts with a suitable number of illumination points. However, how to design these points during measurement remains a challenge. To address this, we developed an imaging technique based on a reinforcement learning in machine learning (ML). This ML approach adaptively feeds back "optimal" illumination pattern during the measurement to detect the existence of specific characteristics of interest, allowing faster measurements while guaranteeing discrimination accuracy. Using a set of Raman images of human follicular thyroid and follicular thyroid carcinoma cells, we showed that our technique requires 3,333 to 31,683 times smaller number of illuminations for discriminating the phenotypes than raster scanning. To quantitatively evaluate the number of illuminations depending on the requisite discrimination accuracy, we prepared a set of polymer bead mixture samples to model anomalous and normal tissues. We then applied a home-built programmable-illumination microscope equipped with our algorithm, and confirmed that the system can discriminate the sample conditions with 104 to 4,350 times smaller number of illuminations compared to standard point illumination Raman microscopy. The proposed algorithm can be applied to other types of microscopy that can control measurement condition on the fly, offering an approach for the acceleration of accurate measurements in various applications including medical diagnosis.

Raman micro-spectroscopy as a tool to study immunometabolism.

In the past two decades, immunometabolism has emerged as a crucial field, unraveling the intricate molecular connections between cellular metabolism and immune function across various cell types, tissues, and diseases. This review explores the insights gained from studies using the emerging technology, Raman micro-spectroscopy, to investigate immunometabolism. Raman micro-spectroscopy provides an exciting opportunity to directly study metabolism at the single cell level where it can be combined with other Raman-based technologies and platforms such as single cell RNA sequencing. The review showcases applications of Raman micro-spectroscopy to study the immune system including cell identification, activation, and autoimmune disease diagnosis, offering a rapid, label-free, and minimally invasive analytical approach. The review spotlights three promising Raman technologies, Raman-activated cell sorting, Raman stable isotope probing, and Raman imaging. The synergy of Raman technologies with machine learning is poised to enhance the understanding of complex Raman phenotypes, enabling biomarker discovery and comprehensive investigations in immunometabolism. The review encourages further exploration of these evolving technologies in the rapidly advancing field of immunometabolism.

Transient stimulated Raman scattering spectroscopy and imaging

Stimulated Raman scattering (SRS) has been developed as an essential quantitative contrast for chemical imaging in recent years. However, while spectral lines near the natural linewidth limit can be routinely achieved by state-of-the-art spontaneous Raman microscopes, spectral broadening is inevitable for current mainstream SRS imaging methods. This is because those SRS signals are all measured in the frequency domain. There is a compromise between sensitivity and spectral resolution: as the nonlinear process benefits from pulsed excitations, the fundamental time-energy uncertainty limits the spectral resolution. Besides, the spectral range and acquisition speed are mutually restricted. Here we report transient stimulated Raman scattering (T-SRS), an alternative time-domain strategy that bypasses all these fundamental conjugations. T-SRS is achieved by quantum coherence manipulation: we encode the vibrational oscillations in the stimulated Raman loss (SRL) signal by femtosecond pulse-pair sequence excited vibrational wave packet interference. The Raman spectrum was then achieved by Fourier transform of the time-domain SRL signal. Since all Raman modes are impulsively and simultaneously excited, T-SRS features the natural-linewidth-limit spectral line shapes, laser-bandwidth-determined spectral range, and improved sensitivity. With ~150-fs laser pulses, we boost the sensitivity of typical Raman modes to the sub-mM level. With all-plane-mirror high-speed time-delay scanning, we further demonstrated hyperspectral SRS imaging of live-cell metabolism and high-density multiplexed imaging with the natural-linewidth-limit spectral resolution. T-SRS shall find valuable applications for advanced Raman imaging.

A metabolic map and artificial intelligence-aided identification of nasopharyngeal carcinoma via a single-cell Raman platform.

Nasopharyngeal carcinoma (NPC) is a complex cancer influenced by various factors. This study explores the use of single-cell Raman spectroscopy as a potential diagnostic tool for investigating biomolecular changes associated with NPC carcinogenesis. Seven NPC cell lines, one immortalised nasopharyngeal epithelial cell line, six nasopharyngeal mucosa tissues and seven NPC tissue samples were analysed by performing confocal Raman spectroscopic measurements and imaging. The single-cell Raman spectral dataset was used to quantify relevant biomolecules and build machine learning classification models. Metabolomic profiles were investigated using ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). By generating a metabolic map of seven NPC cell lines, we identified an interplay of altered metabolic processes involving nucleic acids, amino acids, lipids and sugars. The results from spatially resolved Raman maps and UPLC-MS/MS metabolomics were consistent, revealing an increase of unsaturated fatty acids in cancer cells, particularly in highly metastatic 5-8F and poorly differentiated CNE2 cells. The classification model achieved a nearly perfect classification when identifying NPC and non-NPC cells with an ROC-AUC of 0.99 and a value of 0.97 when identifying 13 tissue samples. This study unveils a complex interplay of metabolic network and highlights the potential roles of unsaturated fatty acids in NPC progression and metastasis. This renders further research to provide deeper insights into NPC pathogenesis, identify new metabolic targets and improve the efficacy of targeted therapies in NPC. Artificial intelligence-aided analysis of single-cell Raman spectra has achieved high accuracies in the classification of both cancer cells and patient tissues, paving the way for a simple, less invasive and accurate diagnostic test.

Visualization of Recrystallization Induced by Ultraviolet Degradation of a Polypropylene Film Using Raman Imaging.

Raman images were constructed for polypropylene (PP) films before and after ultraviolet (UV) irradiation (100 mW, 248-436 nm) for 10 h using several intensity ratios of Raman bands that are sensitive to crystallization of PP. In the images of PP films before the irradiation the intensity ratios are nearly uniform for the films but for those of the PP films after the irradiation, the ratios become large with a mottled pattern, indicating that recrystallization occurs in the PP films upon the irradiation of the UV light. The UV-irradiated PP films show worm-like shaped structures in few micrometer order representing the recrystallization of PP. The temperature gradient of PP is low (273 K), and thus, it is very likely that due to UV energy and polymer fragmentation, PP molecules become more mobile and some parts of molecular chains in amorphous parts of PP molecules lead to their rearrangement and recrystallization. In this study, we demonstrate that Raman imaging clearly detects subtle changes in the crystallinity with a micrometer order structure which morphological images cannot observe.

Additive-rejuvenated anions (De)intercalation into graphite cathode enables optimum dual-ion battery

Cathode-electrolyte interphase (CEI) has particular function in maintaining electrolyte and cathode stability. However, the CEI will also cause some negative effects on ion transport. It is crucial for optimizing the structure of CEI through additives in the electrolyte to improve the electrode reaction. In this study, we proposed using C3H3FO3 (FEC) as an additive to optimize an aqueous LiTFSI (bistrifluoromethanesulfonimide lithium salt) electrolyte. The incorporation of FEC modified the solvation structure of TFSI–, inhibit the formation of CEI with thicker size, thereby facilitating the intercalation of TFSI– into the selected graphite cathode. Consequently, the graphite's discharge capacity was doubled, reaching 47 mAh g−1. The influence of TFSI– solvation structure on CEI formation was further studied by the emerging correlative SEM, Raman imaging and TOF-SIMS techniques and theoretical calculation techniques. Using this optimization strategy, we successfully constructed an aqueous dual-ion battery using C24H10N2O4 and graphite as the anode and cathode with an impressive potential window of 2.55 V, which delivered the energy density of 66 Wh kg−1 at the power density of 128 W kg–1.

Aesthetic Cellulose Filaments with Water-Triggered Switchable Internal Stress and Customizable Polarized Iridescence Toward Green Fashion Innovation.

Healthy, convenient, and aesthetic hair dyeing and styling are essential to fashion trends and personal-social interactions. Herein, we fabricate green, scalable, and aesthetic regenerated cellulose filaments (ACFs) with customizable iridescent colors, outstanding mechanical properties, and water-triggered moldability for convenient and fashionable artificial hairdressing. The fabrication of ACFs involves cellulose dissolution, cross-linking, wet-spinning, and nanostructured orientation. Notably, the cross-linking strategy endows the ACFs with significantly weakened internal stress, confirmed by monitoring the offset of the C-O-C group in the cellulose molecular chain with Raman imaging, which ensures a tailorable orientation of the nanostructure during wet stretching and tunable iridescent polarization colors. Interestingly, ACFs can be tailored for three-dimensional shaping through a facile water-triggered adjustable internal stress: temporary shaping with low-level internal stress in the wet state and permanent shaping with high-level internal stress in the dry state. The health, convenience, and green aesthetic filaments show great potential in personal wearables.

Synthesis of innovative epoxy resin and polyamine hardener microcapsules and their age monitoring by confocal Raman imaging

AbstractWe present an innovative breakthrough encompassing synthesis, characterization, and age monitoring of epoxy resin and polyamine hardener microcapsules to form a dual‐component system for self‐healing anticorrosion coatings. The epoxy resin microcapsules (ER‐MC) are fabricated through oil–water emulsion and in situ polymerization, with poly (urea‐formaldehyde‐melamine) as shell material and a mixture of epoxy resin and n‐butyl glycidyl ether as core material. Modified aliphatic fast reactive polyamine hardener is microencapsulated with polymethylmethacrylate as shell using double emulsion and solvent evaporation method (PAH‐MC). Characterization includes optical microscopy, scanning electron microscopy, and particle size analyzer utilizing laser diffraction. The resulting microcapsules exhibited spherical shape with smooth surface devoid of porosity. Fourier transform infrared spectroscopy‐attenuated total reflection is utilized to confirm encapsulation by identifying the chemical structures of both microcapsule constituents. The thermogravimetric analysis further confirms the core content of microcapsules obtained from solvent extraction. ER‐MC contains approximately 66 wt %, while PAH‐MC stands 40 wt %, both showcasing remarkable thermal stability below 200°C. A ten‐month storage period fails to diminish the presence of the healing agents encapsulated within the polymeric walls, which is revealed by Raman spectroscopy utilizing 2D compositional mapping. This research demonstrates the viability of creating dual microcapsules and exemplify their potential for developing self‐healing coatings with dual‐component film‐forming healing agents.

Spontaneous Raman bioimaging – Looking to 2050

Raman imaging has been employed for a wide range of biological sample analyses, is often chosen for its non-invasiveness, and ability to provide rich information from samples with minimal preparation requirements. In this paper we give a brief overview of the applications of spontaneous Raman imaging in bioanalysis and then consider what Raman imaging in 2050 might look like. We discuss the state of Raman imaging around its inception, then provide a snapshot of current technology, then look towards 2050. We then discuss some of the potential bottlenecks for the continuing development of Raman imaging for biological sample analysis and, where appropriate, outline approaches to overcome these challenges.

Visualization of isothermal crystallization and phase separation in poly[(R)-3-hydroxybutyrate]/poly(L-lactic acid) by low-frequency Raman imaging

The visualization of the variation of the inter/intra molecular interaction (C = O⋯CH3) between poly[(R)-3-hydroxybutyrate] (PHB) and poly-L-lactic acid (PLLA) in the PHB/PLLA miscible blend during phase separation and crystallization process was successfully investigated using Raman imaging. Images of the blend were developed using high- and low-frequency Raman spectra acquired during the isothermal crystallization of the blend, and both of them were compared. The low-frequency region allowed to observe the changes in the hydrogen bonds between the molecular chains in the blend during phase separation and crystallization via a band at 75 cm−1 derived from PHB. The imaging results obtained using the band at 75 cm−1 due to hydrogen bonding (C = O⋯CH3) between molecular chains were in good agreement with the results obtained using the C = O stretching band at 1720 cm−1. Herein, we demonstrated that the low-frequency region of the Raman spectrum is more sensitive to detecting the start of the phase separation and crystallization of PHB than the corresponding high-frequency region.

Degradation selectivity for bamboo fiber and parenchyma lignin-carbohydrates complexes (LCC) esters

The degradation of lignin-carbohydrate complex (LCC) esters has been proven to be crucial for the selective separation of lignocellulosic components. This study utilized Raman microspectroscopy to image the preferential degradation of lignin and LCC esters from the bamboo wall during successive NaOH (0.2 to 5.0 % w/w), H2SO4 (1 to 8 % v/v), and NaClO2 (5 to 20 min) treatments. Raman imaging showed that lignin and LCC esters were selectively removed from the middle lamella of fibers and the secondary wall of parenchyma during NaOH and NaClO2 treatments. In contrast, H2SO4 primarily caused the simultaneous removal of lignin and LCC esters from the fiber wall under harsh conditions (8 %), while the middle lamella of parenchyma was less affected, both morphologically and topochemically. Raman spectral analysis indicated that the band intensity at 1605 cm−1 for lignin and at 1173 cm−1 for LCC esters decreased by >87.0 % in the highly lignified parenchyma secondary wall after a 5.0 % NaOH treatment, while the decrease was <67 % in the fiber wall. Interestingly, a strong linear correlation was observed between LCC esters and carbohydrates in the parenchyma (R2 > 0.912). These findings provide important insights into the graded and classified utilization of bamboo resources.