Adipocytes derived from 3T3-L1 cells are a gold standard for analyses of adipogenesis processes and the metabolism of fat cells. A widely used histological and immunohistochemical staining and mass spectrometry lipidomics are mainly aimed for examining lipid droplets (LDs). Visualizing other cellular compartments contributing to the cellular machinery requires additional cell culturing for multiple labeling. Here, we present the localization of the intracellular structure of the 3T3-L1-derived adipocytes utilizing vibrational spectromicroscopy, which simultaneously illustrates the cellular compartments and provides chemical composition without extensive sample preparation and in the naïve state. Both vibrational spectra (FTIR—Fourier transform infrared and RS—Raman scattering spectroscopy) extended the gathered chemical information. We proved that both IR and RS spectra provide distinct chemical information about lipid content and their structure. Despite the expected presence of triacylglycerols and cholesteryl esters in lipid droplets, we also estimated the length and unsaturation degree of the fatty acid acyl chains that were congruent with known MS lipidomics of these cells. In addition, the clustering of spectral images revealed that the direct surroundings around LDs attributed to lipid-associated proteins and a high abundance of mitochondria. Finally, by using quantified markers of biomolecules, we showed that the fixative agents, paraformaldehyde and glutaraldehyde, affected the cellular compartment differently. We concluded that PFA preserves LDs better, while GA fixation is better for cytochromes and unsaturated lipid analysis. The proposed analysis of the spectral images constitutes a complementary tool for investigations into the structural and molecular features of fat cells.

The full understanding of molecular mechanisms of cell differentiation requires a holistic view. Here we combine label-free FTIR and Raman hyperspectral imaging with data mining to detect the molecular cell composition enabling noninvasive monitoring of cell differentiation and identifying biochemical heterogeneity. Mouse adipose-derived mesenchymal stem cells (AD-MSCs) undergoing adipogenesis were followed by Raman and FT-IR imaging, Oil Red, and immunofluorescence. A workflow of the data analysis (IRRSmetrics4stem) was designed to identify spectral predictors of adipogenesis and test machine-learning (ML) methods (hierarchical clustering, PCA, PLSR) for the control of the AD-MSCs differentiation degree. IRRSmetrics4stem provided insights into the chemism of adipogenesis. With single-cell tracking, we established IRRS metrics for lipids, proteins, and DNA variations during AD-MSCs differentiation. The over 90% predictive efficiency of the selected ML methods proved the high sensitivity of the IRRS metrics. Importantly, the IRRS metrics unequivocally recognize a switch from proliferation to differentiation. This study introduced a new bioassay identifying molecular markers indicating molecular transformations and delivering rapid and machine learning-based monitoring of adipogenesis that can be relevant to other differentiation processes. Thus, we introduce a novel, rapid, machine learning-based bioassay to identify molecular markers of adipogenesis. It can be relevant to identification of differentiation-related molecular processes in other cell types, and beyond the cell differentiation including progression of different cellular pathophysiologies reconstituted in vitro.

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.

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.