Classic infrared (IR) microscopy is limited by the diffraction limit, which obscures subcellular heterogeneity, and by the complex overlap of vibrational bands, which complicates precise molecular assignment. This study presents a comprehensive "IR map of the cell" that provides a standardized framework for label-free chemical identification of subcellular compartments across various human cell lines. By integrating Fourier transform infrared (FTIR) spectroscopy with submicron-resolution optical photothermal infrared (OPTIR) microscopy (∼0.3 μm), the cellular landscape was mapped with improved spatial specificity beyond that achievable by conventional FTIR imaging. Advanced chemometric tools were employed to segment the nucleus, cytoplasm, and regions rich in lipids and glycogen, each characterized by a definitive "IR barcode". Furthermore, in silico modeling validated spectral assignments by simulating cellular fingerprints from reference biocompounds, while detailed spectroscopic characterization of subcellular compartments defined marker bands for proteins, lipids, carbohydrates, and nucleic acids. The modeling supported the interpretation that experimental spectra can be approximated as a linear combination of biomolecular classes, helping to constrain spectral band overlap and refine the proposed "IR barcode" for cellular identification. The developed IR map provides a robust, standardized foundation for the label-free interpretation of cellular chemistry. This study demonstrates the utility of IR microscopy as a powerful diagnostic and analytical tool for monitoring metabolic shifts and cellular status at the micrometric level.

Understanding why some leukemia cells undergo apoptosis upon venetoclax (VEN) treatment while others remain refractory is essential for improving therapeutic outcomes. Here, we integrate Raman microscopy, FT-IR imaging, and chemometrics to explore treatment-associated spectroscopic patterns linked to differential VEN response in Philadelphia-positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL). Using two BCR-ABL1-positive cell lines with intrinsic differences in VEN sensitivity (BV-173, sensitive; SD-1, resistant), we combined classical biological readouts of apoptosis with multimodal vibrational profiling to characterize global biochemical changes induced by VEN. Sensitive BV-173 cells displayed spectroscopic changes consistent with apoptotic progression, reflected in coordinated alterations in nucleic acids, proteins, and lipids. In contrast, resistant SD-1 cells exhibited a distinct treatment-associated biochemical response profile consistent with nonapoptotic adaptation. Spectroscopic fingerprints enabled differentiation between apoptotic and nonapoptotic response patterns in the cell-line models studied. Our findings demonstrate that vibrational spectroscopy, integrated with chemometrics, may provide a complementary analytical framework for exploratory characterization of treatment-associated biochemical phenotypes in leukemia.

Ruxolitinib (RUX), a selective JAK1/JAK2 inhibitor, is considered a therapeutic option for childhood B-cell precursor acute lymphoblastic leukemia (B-ALL) with JAK2 gain-of-function mutations. This study aimed to evaluate whether Raman spectroscopy combined with chemometric analysis can monitor the biochemical effects of RUX treatment in B-ALL cell lines. We employed single-cell confocal Raman imaging, flow cytometry, and Western blotting to assess the response of JAK2-mutated (MUTZ-5 and MHH-CALL-4) and wild-type (SEM) B-ALL cells to 10 μM RUX treatment over 48 h. Dimensionality reduction methods (PCA, t-SNE) and classification approach (o-PLS-DA) were applied to the spectral data to identify treatment-induced changes. RUX selectively reduced STAT5 phosphorylation and induced distinct Raman spectral shifts in JAK2-mutant cells, particularly in DNA- and protein-related bands. No significant changes were observed in JAK2 wild-type cells. The results demonstrate that Raman spectroscopy, when integrated with multivariate analysis, enables the non-destructive tracking of leukemia cell responses to targeted therapy and may support the development of phenotyping tools for drug monitoring in precision oncology.

Type 2 diabetes (T2D) is driven by pancreatic β-cell failure, often in the metabolic context of overweight, obesity, and systemic insulin resistance. Importantly, the accumulation of excess lipids in the pancreas accelerates mitochondrial dysfunction and imbalanced dynamics, both of which are considered early indicators of T2D. The availability of the fatty acid pool determines the composition of cardiolipins (CLs), signature mitochondrial phospholipids. Stearoyl-coenzyme A desaturase 1 (SCD1) remains the lynchpin metabolic enzyme that is responsible for the equipoise biosynthesis of monounsaturated fatty acyl moieties, thereby affecting the overall rate of β-cell survival. The present study investigated the molecular effect of SCD1 depletion on the regulation of mitochondrial energetic status and architecture in pancreatic β-cells that underwent lipotoxic insult. The ablation of SCD1 activity led to severe impairments in mitochondrial bioenergetics, indicated by compromised mitochondrial membrane potential and lower adenosine triphosphate production in opposition to INS-1E cells that were independently subjected to palmitotoxicity. This effect occurred alongside higher amounts of compromised mitochondria and alterations of CL-linked oxidative phosphorylation complexes. The β-cell and pancreatic islet fraction of CL was enriched in 16:1, 18:1, 18:2, 20:4, and 20:3n-6 fatty acids. Such lipid rearrangements coincided with abnormal CL concentrations, alterations of the abundance of CL remodeling or fatty acyl moiety distribution, and cristae shaping effectors. Increases in hallmarks of lipid peroxidation and accelerated susceptibility to ferroptosis complemented observations of the palmitate-mediated collapse of cristae microarchitecture upon SCD1 deficiency. These findings disentangle the yet uncharacterized role of Δ⁹-desaturation in counteracting lipotoxicity-derived mitochondrial decay that occurs during the gradual failure of pancreatic β-cells in T2D by integrating CL acyl-side chain composition and mitochondrial homeostasis.

Since their approval, tyrosine kinase inhibitors (TKIs) have been widely used in antitumor therapy for chronic myeloblastic leukemia. Despite being approved by the FDA in 2001 to treat a rare cancer called chronic myeloid leukemia (CML), imatinib and other TKIs remain subjects of research for several reasons, such as their long-term effects, resistance, or molecular mechanisms. This study uses Raman and fluorescence imaging to investigate the in vitro cytotoxic effects of two TKIs, imatinib and dasatinib, on human aortic endothelial cells (HAECs). A comprehensive range of concentrations for these TKIs was applied to assess their cytotoxic impact based on viability, inflammation, and biochemical profile. Detailed data analysis revealed alterations in the biochemical profiles of cellular components, even though the viability of HAECs was around 80-90%. These changes indicate that, despite the cells retaining viability, they are experiencing considerable sub-lethal stress. Specifically, cells exposed to clinically relevant TKI concentrations showed increased signals from proteins and saturated lipids alongside decreased signals from nucleic acids, cytochromes, and unsaturated lipids. The subcellular analysis highlighted prominent changes in the perinuclear area, dominated by the endoplasmic reticulum and the cytoplasm. These findings suggest that TKIs are cytotoxic to vascular endothelium at concentrations close to those that are clinically observed. The predominant mechanism appears to involve oxidative stress-mediated inflammation, as evidenced by increased lipid content in treated cells and ICAM-1 staining. This cytotoxicity may contribute to the cardiotoxic effects observed during TKI therapy.

Real-time observation of cellular transformations at the subcellular level provides insight into the dynamic biochemical processes underlying cell function and disease. However, many challenges remain in developing methods to analyze metabolic transformations in cells quantitatively. Our study applies Raman spectroscopy (RS) and stimulated Raman scattering (SRS) to link single-molecule resolution with cellular-scale lipid turnover tracking. Using HL-60 cells as a model, we monitor the uptake kinetics of deuterated palmitic acid (dPA), a marker of de novo lipogenesis, from the first minutes to hours. SRS enables microscopic, high-temporal-resolution visualization of dPA incorporation across entire cell volumes, capturing both its distribution and uptake rate. Complementary RS measurements identify distinct metabolic phases: rapid incorporation into pre-existing lipid droplets (LDs) followed by the emergence of new, partially unsaturated LDs associated with cytochrome-linked metabolism. Statistical and spectral analyses reveal cell-to-cell heterogeneity, emphasizing the complexity of lipid metabolism at both the cellular and subcellular levels. By integrating SRS and RS data into time-dependent kinetic profiles, we establish a unified submicron-to-cellular model of fatty acid metabolism. This multi-resolution spectroscopic approach demonstrates how real-time, label-based tracking can uncover dynamic metabolic heterogeneity, advancing understanding of the fatty acid uptake and metabolism in the microscale. ### Competing Interest Statement The authors have declared no competing interest. Foundation for Polish Science, https://ror.org/048zd9m77, POIR.04.04.00-00-16ED/18-00, Start 006.2025 National Science Centre, https://ror.org/03ha2q922, 2023/07/X/ST4/01573, 2024/53/B/ST4/02698 Jagiellonian University

Altered differentiation of blood cell precursors and their clonal expansion occurs in various types of leukemia. One treatment strategy is to induce differentiation into the mature form, which is capable of undergoing apoptosis. It has been found that low doses of doxorubicin (DOX) induce phenotypic and morphological changes in malignant erythroid precursors to erythrocyte-like cells. These are usually studied by time-consuming examination of hemoglobinisation by benzidine staining or glycophorin A expression by Western blot or flow cytometry. As an alternative, we propose an in-depth investigation of DOX-induced erythroid differentiation using Raman spectroscopy and stimulated Raman scattering microscopy, and fast and sensitive classification using the probe for mitochondrial imaging (MitoBADY). Machine learning methods, including Orthogonal Partial Least Squares, Principal Component Analysis, and Multivariate Curve Resolution-Alternating Least Squares, etc., were implemented to extract spectroscopic markers of differentiation from both single spectra and the hyperspectral images.

The aim was to evaluate the potential of MitoBADY as a Raman sensor in monitoring the drug-induced differentiation of promyelocytic cells into neutrophils. Neutrophils are important immune system components, and their metabolic disorders lead to serious diseases. Primary neutrophils found in the bloodstream are short-lived cells, terminally differentiated, and unable to proliferate. Thus, the characteristics of induced differentiation of promyelocytic cells are valuable for better chemotherapy design, as it is difficult to conduct such studies in vivo or ex vivo in bone marrow environments. Alternatively, a stable in vitro model can be used to analyze the differentiation process, e.g., the HL-60 promyelocytic leukemia cell line. The standard method to confirm the promyelocytic differentiation is immunophenotyping, which is complex and laborious. Here, we propose a simple spectroscopic-based alternative that uses the Raman signal of the MitoBADY sensor to identify neutrophil-like cells. We found that the ratio of the intensity of three bands, i.e., A= (I-750+I-2220)/I-750, B=(I-2850+I-2220)/I-2850,I- and A/B, of the Raman spectra of cells incubated with the MitoBADY is a specific marker indicating the induction of neutrophil differentiation of HL-60 cells. Flow cytometry, measuring the level of the expression of the CD11b surface protein, was used as a reference method. Raman measurements allowed the classification of neutrophil-like cells with a sensitivity of 79.8 % and a specificity of 79.5 %. These values demonstrate the great potential of the proposed methodology as a rapid and reliable technique for the evaluation of the differentiation of promyelocytic cells into neutrophils.

Hyperdiploid (HD) B-cell acute lymphoblastic leukemia (ALL) is widely recognized as the most common molecular subtype of leukemia, characterized by the presence of supernumerary chromosomes in the leukemic karyotype. While HD B-ALL is often associated with a favorable prognosis, an important subset of patients still experience relapse, reflecting the biological heterogeneity of this subtype. Current genomic and epigenetic research has shed light on the molecular complexity of HD B-ALL, yet rapid methods for capturing both the metabolic state and the chromosomal content of individual cells remain limited. Here, we introduce a novel Raman spectroscopy (RS)-based approach for the single-cell analysis of HD B-ALL. By detecting characteristic spectroscopic signatures of nucleic acids, proteins, and lipids, RS not only distinguishes malignant cells from normal B cells, but also discriminates between HD B-ALL and other molecular subtypes, including TCF3-PBX1, KMT2A-r, BCR-ABL1, and TEL-AML1. Notably, we developed a partial least-squares regression (PLS-R) model capable of accurately predicting chromosome number from each cell's Raman spectrum, thereby linking molecular fingerprints directly to genomic aberrations. This integrative spectroscopic strategy captures disease heterogeneity and informs therapeutic strategies. Taken together, our proof-of-concept findings highlight RS as a powerful, noninvasive tool for quantifying chromosomal alterations and metabolic phenotypes, adding crucial insights into the complex biology of HD B-ALL and paving the way for broader applications in precision medicine.

Mitotic inhibitors are drugs commonly used in chemotherapy, but their nonspecific and indiscriminate distribution throughout the body after intravenous administration can lead to serious side effects, particularly on the cardiovascular system. In this context, our investigation into the mechanism of the cytotoxic effects on endothelial cells of mitotic inhibitors widely used in cancer treatment, such as paclitaxel (also known as Taxol) and Vinca alkaloids, holds significant practical implications. Understanding these mechanisms can lead to more targeted and less harmful cancer treatments. Human aorta endothelial cells (HAECs) were incubated with selected mitotic inhibitors in a wide range of concentrations close to those in human plasma during anticancer therapy. The analysis of single cells imaged by Raman spectroscopy allowed for visualization of the nuclear, cytoplasmic, and perinuclear areas to assess biochemical changes induced by the drug 's action. The results showed significant changes in the morphology and molecular composition of the nucleus. Moreover, an effect of a given drug on the cytoplasm was observed, which can be related to its mechanism of action (MoA). Raman data supported by fluorescence microscopy measurements identified unique changes in DNA form and proteins and revealed drug-induced inflammation of endothelial cells. The primary goal of mitotic inhibitors is based on the impairment of tubulin formation and the inhibition of the mitosis process. While all three drugs affect microtubules and disrupt cell division, they do so through different MoA, i.e., Vinca alkaloids inhibit microtubule formation, whereas paclitaxel stabilizes microtubules. To sum up, the work shows how a specific drug can interact with endothelial cells.

This work aims to understand better the mechanism of cellular processes accompanying the activation of human T cells and to develop a novel, fast, label-free approach to identify molecular biomarkers for this process. The standard methodology for confirming the activation state of T cells is based on flow cytometry and using antibodies recognizing activation markers. The method provide high specificity detection but may be susceptible to background staining or non-specific secondary antibody reactions. Here, we evaluated the potential of Ramanbased molecular imaging in distinguishing non-activated and activated human T cells. Confocal Raman microscopy was performed on T cells followed by chemometrics to obtain comprehensive molecular information, while Stimulated Raman Scattering imaging was used to quickly provide high-resolution images of selected cellular components of activated and non-activated cells. For the first time, carotenoids, lipids, and proteins were shown to be important biomarkers of T-cell activation. We found that T-cell activation was accompanied by lipid accumulation and loss of carotenoid content. Our findings on the biochemical, morphological, and structural changes associated with activated mature T cells provide insights into the molecular changes that occur during therapeutic manipulation of the immune response. The methodology for identifying activated T cells is based on a novel imaging method and supervised and unsupervised chemometrics. It unambiguously identifies specific and unique molecular changes without the need for staining, fixation, or any other sample preparation.

Metabolism of endothelial cells (ECs) depends on the availability of the energy substrates. Since the endothelium is the first line of defence against inflammation in the cardiovascular system and its dysfunction can lead to the development of cardiovascular diseases, it is important to understand how glucose metabolism changes during inflammation. In this work, glucose uptake was studied in human microvascular endothelial cells (HMEC-1) in high glucose (HG), and additionally in an inflammatory state, using Raman imaging. HG state was induced by incubation of ECs with a deuterated glucose analogue, while the EC inflammation was caused by TNF-α pre-treatment. Spontaneous and stimulated Raman scattering spectroscopy provided comprehensive information on biochemical changes, including lipids and the extent of unsaturation induced by excess glucose in ECs., induced by excess glucose in ECs. In this work, we indicated spectroscopic markers of metabolic changes in ECs as a strong increase in the ratio of the intensity of lipids / (proteins + lipids) bands and an increase in the level of lipid unsaturation and mitochondrial changes. Inflamed ECs treated with HG, revealed enhanced glucose uptake, and intensified lipid production i.a. of unsaturated lipids. Additionally, increased cytochrome c signal in the mitochondrial region indicated higher mitochondrial activity and biogenesis. Raman spectroscopy is a powerful method for determining the metabolic markers of ED which will better inform understanding of disease onset, development, and treatment.

Etravirine (ETV) is an antiretroviral agent that belongs to the class of non-nucleoside reverse transcriptase inhibitors. This study explores the uptake and distribution of ETV in human aortic endothelial cells (HAECs) using Raman spectroscopy combined with chemometrics. The distinctive chemical structure of ETV facilitates tracking of its uptake by observing the Raman band at 2225 cm(-1) in the Raman-silent region. The perinuclear distribution pattern in HAECs depends on drug concentration and incubation time. The uptake of ETV is observed within 5 minutes at a concentration of 10 mu M, as evidenced by Raman images. Lower ETV concentrations, reflective of those found in human plasma, are detectable in HAECs by applying chemometric methods to Raman spectra from the perinuclear region. The ETV accumulation process is crucial in advancing our understanding of the drug's impact on biochemical alterations within endothelial cells. Additionally, ETV emerges as a promising Raman reporter for marking subcellular compartments, leveraging the 2225 cm(-1) band in the cellular Raman silent region. This research contributes valuable insights into the behavior of ETV at the subcellular level, shedding light on its potential applications and impact on subcellular dynamics.

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. Acute lymphoblastic leukaemia (ALL) is a complex disease in pediatric oncology, necessitating accurate diagnostic strategies for effective treatment planning.

Red blood cells (RBCs) play a role in the regulation of vascular tone via release of adenosine triphosphate (ATP) into the vasculature in response to various stimuli. Interestingly, ApoE/LDLR double-deficient (ApoE/LDLR-/-) mice, a murine model of atherosclerosis, display a higher exercise capacity compared to the age-matched controls. However, it is not known whether increased exercise capacity in ApoE/LDLR-/- mice is linked to the altered ATP release from RBCs. In this work, we characterized the ATP release feature of RBCs from ApoE/LDLR-/- mice by exposing them to various stimuli in vitro. The results are linked to the previously reported mechanical and biochemical alterations in RBCs. 3V-induced ATP release from RBCs was at comparable levels for all groups, which indicated that the activity of adenylyl cyclase and the components of upstream signal-transduction pathway were intact. Moreover, hypoxia- and low pH-induced ATP release from RBCs was higher in ApoE/LDLR-/- mice compared to their age-matched controls, a potential contributing factor and a finding in line with the higher exercise capacity. Taken together, augmented hypoxia-induced ATP release from RBCs in ApoE/LDLR-/- mice indicates a possible deterioration in the ATP release pathway. This supports our previous reports on the role of the protein structure alterations of RBC cytosol in hypoxia-induced ATP release from RBCs in ApoE/LDLR-/- mice. Thus, we emphasize that the presented herein results are the first step to future pharmacological modification of pathologically impaired microcirculation.

Chemotherapeutic anthracyclines, like doxorubicin (DOX), are drugs endowed with cytostatic activity and are widely used in antitumor therapy. Their molecular mechanism of action involves the formation of a stable anthracycline-DNA complex, which prevents cell division and results in cell death. It is known that elevated DOX concentrations induce DNA chain loops and overlaps. Here, for the first time, tip-enhanced Raman scattering was used to identify and localize intercalated DOX in isolated double-stranded calf thymus DNA, and the correlated near-field spectroscopic and morphologic experiments locate the DOX molecules in the DNA and provide further information regarding specific DOX-nucleobase interactions. Thus, the study provides a tool specifically for identifying intercalation markers and generally analyzing drug-DNA interactions. The structure of such complexes down to the molecular level provides mechanistic information about cytotoxicity and the development of potential anticancer drugs.

Diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin's lymphoma in adults, is a genetically and metabolically heterogeneous group of aggressive malignancies. The complexity of their molecular composition and the variability in clinical presentation make clinical diagnosis and treatment selection a serious challenge. The challenge is therefore to quickly and correctly classify DLBCL cells. In this work, we show that Raman imaging is a tool with high diagnostic potential, providing unique information about the biochemical components of tumor cells and their metabolism. We present models of classification of lymphoma cells based on their Raman spectra. The models automatically and efficiently identify DLBCL cells and assign them to a given cell-of-origin (COO) subtype (activated B cell-like (ABC) or germinal center B cell-like (GCB)) or, respectively, to a comprehensive cluster classification (CCC) subtype (OxPhos/non-OxPhos). In addition, we describe each lymphoma subtype by its unique spectral profile, linking it to biochemical, genetic, or metabolic features.