DOX-DNA Interactions on the Nanoscale: In Situ Studies Using Tip-Enhanced Raman Scattering.

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.

Raman classification of selected subtypes of acute lymphoblastic leukemia (ALL).

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with chromosome translocations like KMT2A gene rearrangement (KMT2A-r) and BCR-ABL1 fusion gene have been recognized as crucial drivers in both BCP-ALL leukemogenesis and treatment management. Standard diagnostic protocols for proliferative diseases of the hematopoietic system, like KMT2A-r-ALL, are genetically based and strongly molecularly oriented. Therefore, an efficient diagnostic procedure requires not only experienced and multidisciplinary laboratory staff but also considerable instrumentation and material costs. In recent years, a Raman spectroscopy method has been increasingly used to detect subtle chemical changes in individual cells resulting from stress or disease. Therefore, the objective of this study was to identify Raman signatures for the molecular subtypes and to develop a classification method based on the unique spectroscopic profile of in vitro models that represent specific aberrations aimed at KMT2A-r (RS4;11, and SEM) and the BCR-ABL1 fusion gene (SUP-B15, BV-173, and SD-1). Data analysis was based on chemometric methods, i.e. principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and support vector machine (SVM). The PCA-based multivariate model was used for pattern recognition of each investigated group of cells while PLS-DA and SVM were used to build models for the discrimination of spectra from the studied BCP-ALL molecular subtypes. The results showed that the studied molecular subtypes of ALL have characteristic spectroscopic profiles reflecting their peculiar biochemical state. The content of lipids (1600 cm-1), nucleic acids (789 cm-1), and haemoproteins (754, 1130, and 1315 cm-1), which are crucial in cell metabolism, was indicated as the main source of differentiation between subtypes. Identification of spectroscopic markers of cells with BCR-ABL1 or KMT2A-r may be useful in pharmacological studies to monitor the effectiveness of chemotherapy and further to understand differences in molecular responses between leukemia primary cells and cell lines.

What is the ability of inflamed endothelium to uptake exogenous saturated fatty acids? A proof-of-concept study using spontaneous Raman, SRS and CARS microscopy.

Endothelial cells (EC) in vivo buffer and regulate the transfer of plasma fatty acid (FA) to the underlying tissues. We hypothesize that inflammation could alter the functionality of the EC, i.e., their capacity and uptake of different FA. The aim of this work is to verify the functionality of inflamed cells by analyzing their ability to uptake and accumulate exogenous saturated FA. Control and inflammatory human microvascular endothelial cells stimulated in vitro with two deuterium-labeled saturated FA (D-FA), i.e., palmitic (D31-PA) and myristic (D27-MA) acids. Cells were measured both by spontaneous and stimulated Raman imaging to extract detailed information about uptaken FA, whereas coherent anti-Stokes Raman scattering and fluorescence imaging showed the global content of FA in cells. Additionally, we employed atomic force microscopy to obtain a morphological image of the cells. The results indicate that the uptake of D-FA in inflamed cells is dependent on their concentration and type. Cells accumulated D-FA when treated with a low concentration, and the effect was more pronounced for D27-MA, in normal cells, but even more so, in inflamed cells. In the case of D31-PA, a slightly increased uptake was observed for inflamed cells when administered at higher concentration. The results provide a better understanding of the EC inflammation and indicate the impact of the pathological state of the EC on their capacity to buffer fat. All the microscopic methods used showed complementarity in the analysis of FA uptake by EC, but each method recognized this process from a different perspective.

Chloroquine-Induced Accumulation of Autophagosomes and Lipids in the Endothelium.

Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome-lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid composition of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodology based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chemical changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a reference method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concentrations of 1-30 µM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, respectively. The CQ-induced endothelial response was associated with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochemical changes offers a better understanding of the autophagy mechanism in the endothelial cells.

Tunicamycin induced endoplasmic reticulum changes in endothelial cells investigated in vitro by confocal Raman imaging.

This paper describes how tunicamycin (Tu), the most widely used pharmacological agent for inducing endoplasmic reticulum (ER) stress, interacts with endothelial cells. Our results show that tunicamycin enters the cells and accumulates within the ER area. ER stress takes place when improperly folded or damaged proteins begin to accumulate; however, spectroscopic markers of these changes have not been identified as yet. In this work, Raman spectroscopy and scanning electron microscopy imaging of individual endothelial cells treated with Tu were performed. The changes in the biochemical composition of endothelial cells induced by Tu attributed to ER stress were studied in detail. A main feature of the Tu impact on the cells was a decrease of the phospholipid content in the area of ER, and the most abundant lipid with phosphorus groups found there, was identified as sphingomyelin.

FT-IR Hyperspectral Imaging and Artificial Neural Network Analysis for Identification of Pathogenic Bacteria.

Identification of microorganisms by Fourier transform infrared (FT-IR) spectroscopy is known as a promising alternative to conventional identification techniques in clinical, food, and environmental microbiology. In this study we demonstrate the application of FT-IR hyperspectral imaging for rapid, objective, and cost-effective diagnosis of pathogenic bacteria. The proposed method involves a relatively short cultivation step under standardized conditions, transfer of the microbial material onto suitable IR windows by a replica method, FT-IR hyperspectral imaging measurements, and image segmentation by machine learning classifiers, a hierarchy of specifically optimized artificial neural networks (ANN). For cultivation, aliquots of the initial microbial cell suspension were diluted to guarantee single-colony growth on solid agar plates. After a short incubation period when microbial microcolonies achieved diameters between 50 and 300 µm, microcolony imprints were produced by using a specifically developed stamping device which allowed spatially accurate transfer of the microcolonies' upper cell layers onto IR-transparent CaF2 windows. Dry microcolony imprints were subsequently characterized using a mid-IR microspectroscopic imaging system equipped with a focal plane array (FPA) detector. Spectral data analysis involved preprocessing, quality tests, and the application of supervised modular ANN classifiers for hyperspectral image segmentation. The resulting easily interpretable segmentation maps suggest a taxonomic resolution below the species level.

Uptake of fatty acids by a single endothelial cell investigated by Raman spectroscopy supported by AFM.

In this work, confocal Raman imaging was used to study the formation of lipid droplets (LDs) in vitro in a single endothelial cell upon incubation with polyunsaturated fatty acids (10 or 25 µM) including arachidonic acid (AA) and its deuterated analog (AA-d8), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Based on the Raman spectra obtained from a single endothelial cell, it was possible to investigate biochemical changes induced by addition of polyunsaturated fatty acids. In particular, the content of lipids in the formed LDs and the unsaturation degree were identified by Raman spectroscopy by marker bands at 1660 cm-1 due to the C[double bond, length as m-dash]C stretching and at ∼3015 cm-1 due to the stretching mode of [double bond, length as m-dash]C-H associated with C[double bond, length as m-dash]C double bonds (except for a deuterated form where these bands are shifted respectively). To establish if the exogenous fatty acid was taken up by the cell and stored in LDs, a deuterium labelled polyunsaturated fatty acid was used. AA-d8 shows characteristic bands at around 2200-2300 cm-1 assigned to the [double bond, length as m-dash]C-D stretching modes. We established the uptake of AA and the accumulation of EPA into newly formed LDs in the endothelial cells. In contrast, no accumulation of DHA in LDs was observed even though LDs were formed upon DHA incubation. Furthermore, using AFM we demonstrated that the presence of LDs in the endothelium affected endothelial stiffness which could have pathophysiological significance. In summary, the results suggest that the formation of LDs in the endothelium involves exogenous and endogenous polyunsaturated fatty acids, and their relative contribution to the LD formation seems distinct for AA, EPA and DHA.

Diversity among endothelial cell lines revealed by Raman and Fourier-transform infrared spectroscopic imaging.

Growing interest in the role of endothelium under physiological and pathological conditions has led to an increasing demand for its representative in vitro models especially suitable for drug tests and medical diagnostics. There are several endothelial cell lines commercially available whose biochemistry, and hence response to various stimuli, can be different. Recently, two vibrational techniques, Raman and Fourier-transform infrared microscopy, have been found to be potent tools for studying the biochemical composition of a single cell in an easy, rapid and label-free way. However, depending on the applied technique, the results may exhibit some divergence due to different selection rules as well as distinct experimental conditions. This paper presents the methodology of examination and characterization of three popular human endothelial cell lines: HAoEC (primary cells), HMEC-1 and EA.hy926 (immortalized cells). Based on high lateral resolution Raman imaging together with standard and high magnification Fourier-transform infrared measurements, the differences in spectral information and the distribution of biomolecules are presented and discussed.

Raman imaging providing insights into chemical composition of lipid droplets of different size and origin: in hepatocytes and endothelium.

In this work, 3D linear Raman spectroscopy was used to study lipid droplets (LDs) ex vivo in liver tissue and also in vitro in a single endothelial cell. Spectroscopic measurements combined with fluorescence microscopy and/or histochemical staining gave complex chemical information about LD composition and enabled detailed investigations of the changes occurring in various pathological states. Lipid analysis in fatty liver tissue was performed using a dietary mouse model of liver steatosis, induced by a high fat diet (HFD). HFD is characterized by a high percentage of calories from saturated fat (60%) and reflects closely the detrimental effects of dietary habits responsible for increased morbidity due to obesity and its complications in well-developed Western societies. Such diets lead to obesity, hyperlipidemia, insulin resistance, and steatosis that may also be linked to endothelial dysfunction. In the present work, Raman spectroscopy was applied to characterized chemical composition of lipid droplets in hepatocytes from mice fed HFD and in the endothelium treated with exogenous unsaturated free fatty acid (arachidonic acid). The results demonstrate the usefulness of Raman spectroscopy to characterize intracellular lipid distribution in 2D and 3D images and can be used to determine the degree of saturation. Raman spectroscopy shows the potential to be a valuable tool for studying the role of LDs in physiology and pathology. The method is generally applicable for the determination of LDs of different size, origin, and composition. Moreover, for the first time, the process of LD formation in the endothelium was detected and visualized in 3D.

Automatic subtyping of Diffuse Large B-cell Lymphomas (DLBCL): Raman-based genetic and metabolic classification.

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.

Raman microscopy reveals how cell inflammation activates glucose and lipid metabolism.

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.

Raman spectroscopy can recognize the KMT2A rearrangement as a distinct subtype of leukemia.

Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are the two most common hematologic malignancies, challenging to treat and associated with high recurrence and mortality rates. This work aims to identify specific Raman biomarkers of ALL cells with the KMT2A gene rearrangement (KMT2A-r), representing a highly aggressive subtype of childhood leukemia with a poor prognosis. The proposed approach combines the sensitivity and specificity of Raman spectroscopy with machine learning and allows us to distinguish not only myelo- and lymphoblasts but also discriminate B-cell precursor (BCP) ALL with KMT2A-r from other blasts of BCP-ALL. We have found that KMT2A-r ALL cells fixed with 0.5% glutaraldehyde exhibit a unique spectroscopic profile that enables us to identify this subtype from other leukemias and normal cells. Therefore, a rapid and label-free method was developed to identify ALL blasts with KMT2A-r based on the ratio of the two Raman bands assigned to phenylalanine - 1040 and 1008 cm-1. This is the first time that a particular group of leukemic cells has been identified in a label-free way. The identified biomarker can be used as a screening method in diagnostic laboratories or non-reference medical centers.

Alterations in lipid metabolism accompanied by changes in protein and carotenoid content as spectroscopic markers of human T cell activation.

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 Raman-based 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.

Secondary structure of proteins analyzed ex vivo in vascular wall in diabetic animals using FT-IR spectroscopy.

In recent years many methods for ex vivo tissue analysis or diagnosis of diseases have been applied, including infrared absorption spectroscopy. Fourier-transform infrared (FT-IR) absorption microspectroscopy allows the simultaneous monitoring of the content of various chemical compounds in tissues with both high selectivity and resolution. Imaging of tissue samples in very short time can be performed using a spectrometer equipped with a Focal Plane Array (FPA) detector. Additionally, a detection of minor components or subtle changes associated with the functional status of a tissue sample is possible when advanced methods of data analysis, such as chemometric techniques, are applied. Monitoring of secondary structures of proteins has already proved to be useful in the analysis of animal tissues in disease states. The aim of this work was to build a mathematical model based on FT-IR measurements for the prediction of alterations in the content of secondary structures of proteins analyzed by FT-IR in the vascular wall of diabetic animals. For that purpose a spectral database of proteins of known crystallography and secondary structures was assembled. Thirty-seven proteins were measured by means of two FT-IR techniques: transflection and Attenuated Total Reflectance (ATR). The obtained model was tested on cross-sections of rat tail, for which the content of proteins and their secondary structures was well characterized. Then, the model was applied for the detection of possible alterations in the secondary structures of proteins in the vascular wall of diabetic rats and mice. The obtained results suggest a prominent increase in E- and S-structures and a decrease in the content of H-structures in the vascular wall from diabetic mice and rats. FT-IR-based studies of secondary structures of proteins may be a novel approach to study complex processes ongoing in the vascular wall. The obtained results are satisfactory; however, the existing limitations of the method are also discussed.

3D confocal Raman imaging of endothelial cells and vascular wall: perspectives in analytical spectroscopy of biomedical research.

Raman imaging was used to illustrate heterogeneity of a single endothelial cell and the vascular wall sample. The spectral analysis allowed for exploring the complexity of the studied systems in three dimensions and defining the size, volume, shape and biochemical composition of cellular organelles. The ability to construct the 3D maps by a method that does not disrupt the spatial integrity of the cell provided a unique insight into biochemical architecture and cellular processes of endothelium and vascular wall. 3D Raman imaging may be considered as a new trend in analytical spectroscopy to be applied in biomedical research. This method has a potential to be used for medical imaging and diagnostic purposes together with other, already established, imaging techniques.

Stearoyl-CoA desaturase 1 deficiency exacerbates palmitate-induced lipotoxicity by the formation of small lipid droplets in pancreatic ß-cells.

The accelerating accumulation of surplus lipids in the pancreas triggers structural and functional changes in type 2 diabetes-affected islets. Pancreatic ß-cells exhibit a restricted capacity to store fat reservoirs in lipid droplets (LDs), which act as transient buffers to prevent lipotoxic stress. With the increasing incidence of obesity, growing interest has been seen in the intracellular regulation of LD metabolism for ß-cell function. Stearoyl-CoA desaturase 1 (SCD1) is critical for producing unsaturated fatty acyl moieties for fluent storage into and out of LDs, likely affecting the overall rate of ß-cell survival. We explored LD-associated composition and remodeling in SCD1-deprived INS-1E cells and in pancreatic islets in wildtype and SCD1-/- mice in the lipotoxic milieu. Deficiency in the enzymatic activity of SCD1 led to decrease in the size and number of LDs and the lower accumulation of neutral lipids. This occurred in parallel with a higher compactness and lipid order inside LDs, followed by changes in the saturation status and composition of fatty acids within core lipids and the phospholipid coat. The lipidome of LDs was enriched in 18:2n-6 and 20:4n-6 in ß-cells and pancreatic islets. These rearrangements markedly contributed to differences in protein association with the LD surface. Our findings highlight an unexpected molecular mechanism by which SCD1 activity affects the morphology, composition and metabolism of LDs. We demonstrate that SCD1-dependent disturbances in LD enrichment can impact pancreatic ß-cells and islet susceptibility to palmitate, which may have considerable diagnostic and methodological value for the characterization of LDs in human ß-cells in type 2 diabetes patients.

Investigation of the Immunogenic Properties of Ovalbumin Modified by Urban Airborne Particulate Matter.

Exposure to air particulate matter (PM) is linked to the blood oxidative stress and systemic inflammation. The aim of this study was to elucidate whether oxidative PM modification of ovalbumin (OVA), the major antioxidant serum protein, may alter its antigenicity and/or immunogenicity. Ovalbumin was exposed via dialysis to the standard urban PM (SRM 1648a) or to PM with removed organic content (encoded as LAP). Both structural changes and biological properties of PM-modified OVA were measured. T lymphocytes and dendritic cells (the major antigen-presenting cells) isolated from C57BL/6 and OT-II (323-339 epitope) OVA-specific T cell receptor (TCR)-transgenic mice were used to test the effect of PM on OVA immunogenicity. The immunogenicity of both SRM 1648a and LAP-modified OVA was significantly higher than that of control OVA, as measured by the epitope-specific T cell proliferation and interferon γ production by the stimulated cells. This effect was associated with mild oxidative changes in the carrier molecule outside the structure of the OVA epitope and with increased resistance to proteolysis of PM-modified OVA. Interestingly, dendritic cells showed enhanced capacity for the uptake of proteins when the cells were cultured with PM-modified OVA. Our results suggest that the enhanced immunogenicity of PM-modified OVA is not associated with altered antigenicity or antigen presentation. However, it may result from slower degradation and longer persistence of modified antigens in dendritic cells. Whether this phenomenon is associated with enhanced risk prevalence of autoimmune diseases observed in the areas with high urban PM pollution needs to be explained.

Reliable cell preparation protocol for Raman imaging to effectively differentiate normal leukocytes and leukemic blasts.

Leukemias are a remarkably diverse group of malignancies originating from abnormal progenitor cells in the bone marrow. Leukemia subtypes are classified according to the cell type that has undergone neoplastic transformation using demanding and time-consuming methods. Alternative is Raman imaging that can be used both for living and fixed cells. However, considering the diversity of leukemic cell types and normal leukocytes, and the availability of different sample preparation protocols, the main objective of this work was to verify them for leukemia and normal blood cell samples for Raman imaging. The effect of glutaraldehyde (GA) fixation in a concentration gradient (0.1 %, 0.5 %, and 2.5 % GA) on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs) was verified. Changes in the secondary structure of proteins within cells were indicated as the main effect of fixation, as shown by an increase in band intensity at 1041 cm-1, characteristic for in-plane δ(CH) deformation in phenylalanine (Phe). Different sensitivity of mononuclear and leukemic cells to fixation was observed. While the 0.1 % concentration of GA was too low to preserve the cell structure for an extended period of time, a GA concentration of 0.5 % seemed optimal for both normal and malignant cells. Chemical changes in PBMCs samples stored for 11 days were also investigated, which manifested in numerous modifications in the secondary structure of proteins and the content of nucleic acids. The impact of cell preculturing for 72 h after unbanking was verified, and there was no significant effect on the molecular structure of cells fixed with 0.5 % GA. In summary, the developed protocol for the preparation of samples for Raman imaging allows for the effective differentiation of fixed normal leukocytes from malignant T lymphoblasts.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy of a single endothelial cell.

Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) with the use of a slide-on germanium accessory followed by chemometric analysis allowed for providing meaningful information about the biochemical composition of a single endothelial cell. In this work, the methodology of the ATR-FTIR measurements of dried cells and dried cells immersed in water solution is presented. The contact of the cell and Ge crystal was set up manually and monitored through the integration of the amide I band. Additionally, the cell imaging in transreflection mode was tested, but the spectral differences between sub-cellular structures were not prominent in the registered spectra. It has been shown that the ATR-FTIR method gives better results due to the increased spatial resolution and S/R ratio as well as small contribution of the optical artifacts.

Raman and fluorescence imaging of phospholipidosis induced by cationic amphiphilic drugs in endothelial cells.

Cationic amphiphilic drugs (CADs) are known from lysosomotropism, drug-induced phospholipidosis (DIPL), activation of autophagy, and decreased cell viability, but the relationship between these events is not clear and little is known about DIPL in the endothelium. In this work, the effects of fluoxetine, amiodarone, clozapine, and risperidone on human microvascular endothelial cells (HMEC-1) were studied using a combined methodology of label-free Raman imaging and fluorescence staining. Raman spectroscopy was applied to characterize biochemical changes in lipid profile and their distribution in the cellular compartments, while fluorescence staining (LysoTracker, LipidTOX, LC3B, and JC-1) was used to analyze lysosome volume expansion, activation of autophagy, lipid accumulation, and mitochondrial membrane depolarization. We demonstrated that fluoxetine, amiodarone, and clozapine, but not risperidone, at non-toxic concentrations induced lipid accumulations in the perinuclear and cytoplasmic regions of endothelial cells. Spectroscopic markers of DIPL included a robust increase in the ratio (lipid/(protein + lipid)), an increase in choline-containing lipid, fatty acids, and the presence of cholesterol esters, while starvation-induced activated autophagy revealed a spectroscopic signature associated with subtle changes in the lipid profile only. Interestingly, lysosomal volume expansion, occurrence of DIPL, and activation of autophagy induced by selected CADs all depended on drug-accumulation in acidic pH of lysosome cellular compartments whereas reduced endothelial viability did not, and was attributed to mitochondrial mechanisms as evidenced by a decreased mitochondrial transmembrane potential. In conclusion, drug-induced phospholipidosis in the endothelium did not reduce endothelial viability per se and can be efficiently assayed by Raman imaging.