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.

Brain microvascular endothelial cells experience hypoxic conditions in several neurodegenerative disease processes and the underlying mechanisms still need to be explored. Current imaging modalities and biochemical assays require many specific markers that should be detected to identify the hypoxic response, especially at a level of single cells. This study presents a single-cell molecular imaging approach utilizing Fourier-Transform Infrared and Raman spectroscopy. Those methods enable the simultaneous detection of proteins, lipids, and nucleic acids encoded in their unique vibrational fingerprints. By establishing ratiometric estimators, we measured upregulated lipid metabolism, structural changes of proteins and asses DNA:RNA ratio at the single-cell level induced by oxygen depletion. Moreover, this approach allows for analyzing changes within specific cellular compartments, including nuclei, providing a comprehensive understanding of how hypoxia affects cellular functions and metabolism. Our findings pave the way for future investigations into the cellular adaptations to hypoxia in brain endothelial cells, potentially revealing novel therapeutic targets for neurodegenerative diseases.

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.

The blood–brain barrier (BBB) is a coordinated multicellular interface in which intercellular communication shapes responses to injury and metabolic stress. However, the contributions of individual cell types to collective stress adaptation remain poorly understood. Here, we examined molecular and metabolic responses of 2D neurovascular unit (NVU) co-culture models comprising endothelial cells, astrocytes, and pericytes exposed to hypoxia or sublethal HgCl₂ toxicity. An integrated platform combining gene and protein expression profiling, label-free vibrational spectroscopic imaging, and machine-learning-based analysis was used to assess stress-induced changes in cell-cycle regulation and metabolism across mono- and co-culture configurations. Hypoxia broadly suppressed G₁/S and G₂/M regulators, inducing cell-cycle arrest with limited cell death, consistent with an adaptive response. HgCl₂ caused heterogeneous, model-dependent changes in viability and proliferative capacity. Spectroscopic phenotyping revealed that the triple co-culture exhibited spectral features resembling those of pericyte monocultures, although the contributions of cell–cell interactions and differences in culture conditions could not be distinguished. Fluorescence imaging further revealed mitochondria-associated signals at cell–cell contact sites, suggesting a possible association of mitochondrial material with regions of close cellular interaction. The presence of pericytes was associated with a more stress-resistant cellular phenotype in the investigated co-culture configurations. The developed spectroscopic–molecular platform provides a label-free framework for characterizing cellular communication and stress responses in multicellular NVU co-culture models.

The blood–brain barrier (BBB) is a coordinated multicellular interface in which intercellular communication shapes responses to injury and metabolic stress. However, the contributions of individual cell types to collective stress adaptation remain poorly understood. Here, we examined molecular and metabolic responses of 2D BBB models comprising endothelial cells, astrocytes, and pericytes exposed to hypoxia or sublethal HgCl₂ toxicity. An integrated platform combining gene and protein expression profiling, label-free vibrational spectroscopic imaging, and machine-learning-based analysis was used to assess stress-induced changes in cell-cycle regulation and metabolism across monoculture and co-culture configurations. Hypoxia broadly suppressed G1/S and G2/M regulators, inducing cell-cycle arrest with limited cell death, consistent with an adaptive response. HgCl₂ caused heterogeneous, model-dependent changes in viability and proliferative capacity. Spectroscopic phenotyping showed that co-cultured cells acquired molecular profiles characteristic of pericytes, indicating a dominant pericyte-driven influence on the cellular community. Fluorescence imaging further revealed intercellular mitochondrial transfer at cell–cell contact sites. These findings identify pericytes as key contributors to stress resistance and BBB cell survival. The developed spectroscopic–molecular platform provides a label-free framework for monitoring cellular communication and stress responses in multicellular BBB models.