Raman Spectroscopy Imaging Research Articles

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.

Raman spectroscopy of zirconium hydride

The characterization of zirconium hydride is important in the nuclear industry because of the hydrogen-induced embrittlement of Zircaloy cladding and its use as a neutron moderator. This paper introduces the use of Raman spectroscopy for the characterization of zirconium hydride. First-principles density functional theory (DFT) calculations were used to predict the Raman spectra of ζ-ZrH0.5, γ-ZrH, δ-ZrH1.5, δ-ZrH1.66, and ε-ZrH2 with all their predicted symmetries; ζ-ZrH0.5 (P3m1, R3¯m, C2/m, Cm, Cmmm, and Pn3¯m); γ-ZrH (P222, Ccce, and P42/mmc); δ-ZrH1.5 (P4¯m2, P42/mcm, Fmmm, Pn3¯m, Ibam, P2/c, PI, and P42/nnm); δ-ZrH1.66 (Fm3¯m); and ε-ZrH2 (Fm3¯m, R3¯m, and I4/mmm). Two samples of Zircaloy-4 containing 133 wt ppm and 360 wt ppm hydrogen were characterized by Raman spectroscopy, showing two signal lines at 215 cm−1 and 1,187 cm−1, which were assigned to the presence of δ-ZrH1.66. These signals had a good spatial correlation with visible hydride precipitates in Raman spectroscopy images. This work provides the basis for the characterization of all possible zirconium hydride compositions and structures using Raman spectroscopy.

High-throughput broad-spectrum analysis of tetracyclines via surface-enhanced Raman spectroscopy imaging technology

Surface-enhanced Raman spectroscopy (SERS) often results in detection errors and/or omissions because of insufficient specificity and/or unstable signals, thereby limiting broader sensing applications. We synthesized an aptamer with a targeted recognition of tetracyclines (TCs) via clipping, mutation, and fragment addition. High-throughput and broad-spectrum analysis of trace TCs in complex systems was enabled by combining Raman spectroscopy automated platformwith SERS imaging technology,the Au/Ag nanoparticles were used for Raman-enhancing substrates and aptamer recognition. A 0.07-pM TCs detection limit was observed along with selectivity and stability. Detection of TCs in real samples, such as serum and wastewater, was demonstrated. Overall, the TCs detection provides new potential SERS sensing and imaging strategies.

Raman spectroscopic measurements and imaging on sub-newton Berkovich and spherical imprints in fused silica

This paper lays out best practices for evaluating and optimizing a Raman spectroscopy setup to ensure the collection of reliable spectral data and/or Raman images on indented glasses. The Raman spectroscopic measurements and imaging were conducted on residual imprints created with Berkovich and spherical probes at forces in the sub-newton range in fused silica. The capability of a conventional optical instrument for mapping spectral variations in sub-newton imprints on glasses is evaluated by studying the influence of the optical configuration (choice of microscope objective) on the spatial resolution of the spectroscopy setup. The spatial resolution was quantitatively assessed in Z profile measurements and qualitatively evaluated by mapping changes in spectral features and correlated densification within the indented regions of fused silica specimens. The paper discusses the importance of appropriately matching the analysis volume of the Raman spectroscopic setup with the size of the indentation-induced densification zone by demonstrating the detrimental effects a mismatch may have on accurately capturing the magnitude of spectral changes and correlated densification.

Biochemical changes in lipid and protein metabolism caused by mannose-Raman spectroscopy studies.

Biochemical analysis of human normal bronchial cells (BEpiC) and human cancer lung cells (A549) has been performed by using Raman spectroscopy and Raman imaging. Our approach provides a biochemical compositional mapping of the main cell components: nucleus, mitochondria, lipid droplets, endoplasmic reticulum, cytoplasm and cell membrane. We proved that Raman spectroscopy and Raman imaging can distinguish successfully BEpiC and A549 cells. In this study, we have focused on the role of mannose in cancer development. It has been shown that changes in the concentration of mannose can regulate some metabolic processes in cells. Presented results suggest lipids and proteins can be considered as Raman biomarkers during lung cancer progression. Analysis obtained for bands 1444 cm-1, and 2854 cm-1 characteristic for lipids and derivatives proved that the addition of mannose reduced levels of these compounds. Results obtained for protein compounds based on bands 858 cm-1, 1004 cm-1 and 1584 cm-1 proved that the addition of mannose increases the values of protein in BEpiC cells and blocks protein glycolisation in A549 cells. Noticing Raman spectral changes in BEpiC and A549 cells supplemented with mannose can help to understand the mechanism of sugar metabolism during cancer development and could play in the future an important role in clinical treatment.

Visual detection of microplastics using Raman spectroscopic imaging.

As a new type of pollutant in the marine environment and terrestrial ecosystems, microplastics have attracted widespread attention. Assessing the ecological risk of microplastics relies on accurately detecting small-sized particles in the environment. Microplastics exhibit unique "fingerprint" characteristics in Raman spectroscopy, making them suitable for rapid identification. In this study, we achieved visualization of microplastics through pseudo-color images generated by Raman spectroscopy imaging. Pseudo-color imaging maps were generated by selecting characteristic peaks and the classical least-squares fitting method was used to visually represent the distribution of different microplastics. The study explored the potential of Raman spectroscopy and its mapping mode in distinguishing various types of mixed microplastics and demonstrated that this approach can identify microplastics in complex environmental samples. Specifically, a cloud-point extraction followed by membrane filtration method was successfully applied to identifying mixed-component microplastics. In summary, the category, quantity, location, and differentiation of microplastics can be accurately analyzed by Raman spectroscopy, which provides a basis for assessing their ecological risk.

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.

Tailoring a hierarchical porous carbon electrode from carbon black via 3D diatomite morphology control for enhanced electrochemical performance.

Carbon black, a nano-porous material usually derived from the pyrolysis of waste tyres possesses varied particle sizes and morphology making it a viable material for several engineering applications. However, the high tendency for CB to agglomerate remains a challenge. To address this, bio-templating has been employed to produce a nanostructured porous carbon electrode material for supercapacitor applications using diatomite as a template. Diatomite-synthesized activated carbon (DSAC) was fabricated through a three-step process involving acid treatment of diatomite, thermal activation of carbon black, and bio-template synthesis. The resulting material was thoroughly characterized using XRD, Raman spectroscopy, BET analysis, and SEM imaging. Its electrochemical properties were assessed through cyclic voltammetry, galvanostatic charge-discharge, and electrochemical impedance spectroscopy. The DSAC material exhibited a high specific surface area of 266.867 m2 g-1, pore volume of 0.6606 cm3 g-1, and mean pore radius of 1.8943 nm. The electrochemical evaluation revealed that DSAC demonstrates excellent electrochemical performance, achieving a high specific capacitance of 630.18 F g-1 and retaining 94.29% capacitance after 5000 cycles at 1 A g-1. The DSAC electrode is eco-friendly and a promising candidate for supercapacitor applications.

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

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.

Visual whole-process monitoring of pesticide residues: An environmental perspective using surface-enhanced Raman spectroscopy with dynamic borohydride-reduced silver nanoparticles

Environmental monitoring of pesticide residues in crops is essential for both food safety and environmental protection. Traditional methodologies face challenges due to the interference of endogenous compounds in peel and pulp tissues, often being invasive, labor-intensive, and inadequate for real-time observation of hazardous substance distribution. In this study, dynamic borohydride-reduced nanoparticles were employed as enhanced substrates. For the first time, surface-enhanced Raman spectroscopy (SERS) imaging was harnessed to enable whole-process visual detection of pesticide residues. The developed method is both stable and sensitive, boasting a detection lower limit below 1 pg/mL, coupled with robust quantitative analytical capabilities. This technique was successfully employed to detect residue signals across various crops and fruit juices. Furthermore, SERS imaging was utilized to map the distribution of pesticide residues from the exterior to the interior of fruits and vegetables. Vertex component analysis further refined the process by mitigating interference from plant autofluorescence. Collectively, this innovative strategy facilitates comprehensive pesticide residue monitoring, offering a potent tool for controlling hazardous substances in crops. Its potential applications extend beyond food safety, holding significant promise for sustainable agricultural production and enhanced environmental safeguarding.

The Application of Handheld Near-Infrared Spectroscopy and Raman Spectroscopic Imaging for the Identification and Quality Control of Food Products.

The following investigations describe the potential of handheld NIR spectroscopy and Raman imaging measurements for the identification and authentication of food products. On the one hand, during the last decade, handheld NIR spectroscopy has made the greatest progress among vibrational spectroscopic methods in terms of miniaturization and price/performance ratio, and on the other hand, the Raman spectroscopic imaging method can achieve the best lateral resolution when examining the heterogeneous composition of samples. The utilization of both methods is further enhanced via the combination with chemometric evaluation methods with respect to the detection, identification, and discrimination of illegal counterfeiting of food products. To demonstrate the solution to practical problems with these two spectroscopic techniques, the results of our recent investigations obtained for various industrial processes and customer-relevant product examples have been discussed in this article. Specifically, the monitoring of food extraction processes (e.g., ethanol extraction of clove and water extraction of wolfberry) and the identification of food quality (e.g., differentiation of cocoa nibs and cocoa beans) via handheld NIR spectroscopy, and the detection and quantification of adulterations in powdered dairy products via Raman imaging were outlined in some detail. Although the present work only demonstrates exemplary product and process examples, the applications provide a balanced overview of materials with different physical properties and manufacturing processes in order to be able to derive modified applications for other products or production processes.

Feasibility of gold nanocones for collocated tip‐enhanced Raman spectroscopy and atomic force microscope imaging

AbstractMicrocantilever probes for tip‐enhanced Raman spectroscopy (TERS) have a grainy metal coating that may exhibit multiple plasmon hotspots near the tip apex, which may compromise spatial resolution and introduce imaging artefacts. It is also possible that the optical hotspot may not occur at the mechanical apex, which introduces an offset between TERS and atomic force microscope maps. In this article, a gold nanocone TERS probe is designed and fabricated for 638 nm excitation. The imaging performance is compared to grainy probes by analysing high‐resolution TERS cross‐sections of single‐walled carbon nanotubes. Compared to the tested conventional TERS probes, the nanocone probe exhibited a narrow spot diameter, comparable optical contrast, artefact‐free images, and collocation of TERS and atomic force microscope topographic maps. The 1/ spot diameter was 12.5 nm and 19 nm with 638 nm and 785 nm excitation, respectively. These results were acquired using a single gold nanocone probe to experimentally confirm feasibility. Future work will include automating the fabrication process and statistical analysis of many probes.

Raman Spectroscopy Imaging in Evaluation of Immune Cells Activation Status

Introduction: The ability to assess the activation status of immune cells is useful for medical diagnostics and currently also for monitoring the performance of immune cells-based advanced therapy medicinal products (ATMP) such as the chimeric antigen receptor (CAR)-T cells. CAR-T cells numbers and immunophenotype are routinely monitored to avoid poor expansion or exhaustion phenotype. In this study, we utilized Raman spectroscopy (RS) as a label-free, non-destructive method for assessing T cells activation and macrophages polarization (pro-inflammatory M1 vs immunosuppressive M2). RS is a well-defined, non-elastic light scattering-based spectroscopic technique that followed by chemometric analysis provides unique and comprehensive information regarding the molecular structure, metabolic signature and biochemical composition of tissues and cells at subcellular level. To assess if RS would be a valid approach for detection of CAR-T activation we first developed CD19CAR-transduced Jurkat cell line model. Although Jurkat cells, a leukemic T cell line, lack cytolytic activity, they do produce IL-2 and upregulate CD69 upon activation like CAR-T thus serve as a convenient model. Materials and Methods: T cells and monocyte were isolated from healthy donors' blood. T cells were activated with coated CD3/CD28 beads. Jurkat cells were transduced with anti-CD19 chimeric TCR. CAR-J cells activation upon contact with CD19+ cells were confirmed by CD69 expression (flow cytometry) and calcium fluxes (fluorescence microscopy). Monocyte were differentiated to macrophages using CSF1, while THP1 monocytic cell line with PMA. Macrophages were subsequently polarized into M1 and M2 with IL-4 and IL-13 or IFN-gamma and LPS respectively. M0 macrophages or naïve T cells were used as a reference and control. Cell phenotypes were confirmed using flow cytometry, q-PCR, fluorescent confocal microscopy and proliferation assay. Cells were fixed in 0.5% glutaraldehyde and imaged at 533 and 633 nm with a WITec Alpha 300 Raman microscope. Collected data were analyzed by chemometric methods: K-means cluster analysis (KMCA) followed by Partial Least Squares-Discrimination Analysis (PLS-DA) and Principal Component Analysis (PCA). Results: RS analysis of polarized macrophages showed the increased signal from hemoproteins (740-760 cm -1) in all three states. Interestingly we observed different hemoproteins to lipids (1440, 1240, 1660, 2850 cm -1) ratio between M0, M1 and M2. Also degree of lipid unsaturation can be considered an important parameter in the identification of the M1 phenotype of macrophages. Chemometric analyzes revealed significant morphological (increased cell and nucleus sizes) biochemical and difference between activated T lymphocytes and naive cells. Carotenoids present in non-activated T cells disappeared after activation. Conversely, lipids in the form of distinct clusters were observed after activation. Moreover, in the spectra of cell nuclei (733, 790, 1583 cm -1) of activated lymphocytes, less condensed chromatin was manifested by a lower intensity of marker bands. We observed similar changes for activated CAR-Jurkat, though the morphological changes were more subtle and carotenoids were not observed in Jurkat cells. Conclusions: The obtained results indicate the potential utility of Raman spectroscopy methods in identifying lymphocyte states activation and macrophage polarization . Acknowledgements. The studies were performed as a part of the “ Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Window into the mind: Advanced handheld spectroscopic eye-safe technology for point-of-care neurodiagnostic.

Traumatic brain injury (TBI), a major cause of morbidity and mortality worldwide, is hard to diagnose at the point of care with patients often exhibiting no clinical symptoms. There is an urgent need for rapid point-of-care diagnostics to enable timely intervention. We have developed a technology for rapid acquisition of molecular fingerprints of TBI biochemistry to safely measure proxies for cerebral injury through the eye, providing a path toward noninvasive point-of-care neurodiagnostics using simultaneous Raman spectroscopy and fundus imaging of the neuroretina. Detection of endogenous neuromarkers in porcine eyes' posterior revealed enhancement of high-wave number bands, clearly distinguishing TBI and healthy cohorts, classified via artificial neural network algorithm for automated data interpretation. Clinically, translating into reduced specialist support, this markedly improves the speed of diagnosis. Designed as a hand-held cost-effective technology, it can allow clinicians to rapidly assess TBI at the point of care and identify long-term changes in brain biochemistry in acute or chronic neurodiseases.

Noncovalent Dualism in Perylene-Diimide-Based Keggin Anion Complexes: Theoretical and Experimental studies.

We report the synthesis and comprehensive characterization of organic-inorganic hybrid salts formed by bis-cationic N,N'-bis(2-(trimethylammonium)ethylene)perylene-3,4,9,10-tetracarboxylic acid bisimide (PTCD2+) and Keggin-type [XW12O40]n- (X = Si, n = 4; X = P, n = 3) polyoxometalates. (PTCD)3[PW12O40]2·3DMSO·2H2O (2) and (PTCD)2[SiW12O40]·DMSO·2H2O (3) were structurally characterized by single crystal X-ray diffraction. The cations in both structures exhibited infinite chainlike arrangements through π-π interactions, contrasting with the previously reported cation-anion stacking observed in naphthalene diimide derivatives. A detailed theoretical study employing topological analysis of the electron density distribution within the quantum theory of atoms in molecules approach provided further insights into this structural dualism. Atomic force microscopy analyses revealed the formation of self-assembled supramolecular structures on graphite from molecular monolayers (3 nm of thick) to submicrometer aggregates for 2. Hyperspectral Raman spectroscopy imaging revealed that such heterostructures are likely formed by an enhanced π-π interactions. Both complexes demonstrated interesting electrochemical behavior, photoluminescence and X-ray-induced luminescence. Electron spin resonance analysis confirmed charge separation in both compounds, with enhanced efficiency observed in compound 2. Our findings of these perylene-based organic-inorganic hybrid salts offer the potential for their application in optoelectronic devices and functional materials.

Effects of Na/Al ratio on phonon sideband spectra and luminescent characteristics of Eu3+ doped sodium aluminosilicate glasses

In this work, sodium aluminosilicate (NAS) glass systems were fabricated via traditional melting technique with various Na/Al ratios. Structure and surface morphology of the fabricated glasses were investigated by X-ray diffractometer, Raman spectroscopy and SEM images. Optical characteristics of the fabricated glasses were inspected by photoluminescence (PL) and photoluminescence excitation (PLE) spectra, and the observations showed that intensity of the excitation peak of Eu3+ ions in the NAS glasses at 392 nm increases while one at 463 nm decreases with the increasing of Na/Al ratio. Analysis of the phonon sideband spectra shown a change of multiphonon relative rate (Wmp/W0) of the synthesized glasses relates to Na/Al ratio. By applying the Judd-Ofelt theory, Ωλ = 2,4,6 parameters were evaluated from the PL spectra and used these parameters to estimate the radiative parameters like calculated lifetime (τcal), effective line width (Δλeff), transition probability rate (A), emission cross-section (σp) and branching ratio (βR). Decay curves were measured to investigate luminescence quantum efficiency (η) of Eu3+ emission and η gets value in the 53%–65% region. The color coordinates (x, y) and correlated color temperature (CCT) were also estimated for the synthesized NAS glasses.

Analysis of Infrared and Raman Imaging Data Using Alternating and Modified Alternating Least Squares

Modified alternating least squares (MALS) outperforms alternating least squares (ALS) in the analysis of infrared and Raman image spectral data. MALS offers superior stability thanks to ridge regression and a substantial speed advantage due to the kernel nature of the algorithm, reducing computational overhead. MALS excels in resolving basis vectors even in low signal-to-noise, nearly collinear data, whereas ALS often falls short. For spectroscopic imaging, both MALS and other ALS methods rely on spatial resolution between sample components, as low spatial resolution leads to increased mixing of components. Spectroscopic imaging combines spectroscopy and digital imaging to extract chemical composition. Multivariate curve resolution (MCR)’s foundation in ALS regression makes it a vital tool for this analysis, enabling a comprehensive examination of complex spectroscopic images. This tutorial delves into the mathematical techniques necessary for extracting chemical insights from infrared and Raman spectroscopic images. While this discussion focuses on two-dimensional spatial data, the methodology can be extended to three-dimensional data.

Silicon-Based Nanoelectronic Semiconductor Material and Its Surface-Enhanced Raman Image Signals

One-dimensional Zinc Oxide (ZnO) nanomaterials have important applications in devices, such as solar energy, sensors, and piezoelectric materials due to surface effects and quantum size effects. As a semiconductor material, Si has good thermal conductivity and low resistivity. A layer of n-type ZnO nanowires is grown on P-type monocrystalline silicon by transferring the Anodic Aluminum Oxide (AAO) template. The enhanced Raman effect on the surface is attempted by preparing n-type ZnO-P-type Si heterojunction nanostructures. This paper uses Si-based AAO as a template and uses electrochemical deposition method to prepare silicon-based nano-electronic semiconductor material ZnO/AAO/Si. According to SEM analysis, there are uniform pores on the surface of the material, and the cross-section shows that ZnO nanowires have been assembled into the AAO/Si structure. XRD analysis shows that ZnOn nanowires have a hexagonal wurtzite structure. In optoelectronic testing, through PL spectral analysis, the semiconductor material ZnO/AAO/Si has a strong yellow green emission peak near 574 nm. Field emission analysis shows that the β value of the ZnO field enhancement factor in the ZnO/AAO/Si structure is relatively high. The Surface-Enhanced Raman Spectroscopy (SERS) image signal experiment shows that the silicon-based nano-electronic semiconductor material prepared by electrochemical deposition method has good SERS characteristics. The SERS spectral intensity increases with the increase of the concentration of the detected substance and can be used as a SERS substrate to achieve Raman spectral image signal detection of chemical substances with low concentration.

Molecular tracking of interactions between progenitor and endothelial cells via Raman and FTIR spectroscopy imaging: a proof of concept of a new analytical strategy for in vitro research

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24 h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions.