Raman and AFM-IR Spectroscopy in the Characterization of Giant Plasma Membrane Vesicles Isolated from Microglia and Glioblastoma Cells.

Giant plasma membrane vesicles (GPMVs) are widely used as cell-derived models of biological membranes, yet their spectroscopic characterization remains limited. In this work, we propose a preparation and analytical workflow enabling Raman and Atomic Force Microscopy-based infrared (AFM-IR) spectroscopic investigation of GPMVs isolated from microglial (human microglial clone 3, HMC3) and glioblastoma (LN229, U87 MG, and U251 MG) cell lines. Storage conditions and isolation parameters were systematically evaluated to ensure preservation of vesicle morphology and biochemical stability prior to spectroscopic measurements. Raman spectroscopy provided label-free molecular fingerprints of GPMV composition, revealing variations in lipid and protein contributions depending on the cellular origin of the vesicles. Complementary characterization was achieved using AFM-IR spectroscopy, enabling the detection of differences in carbohydrate-, protein-, and lipid-related vibrational bands at the nanoscale, without the need for sample labeling. Chemometric analysis, including principal component analysis and partial least squares discriminant analysis, further confirmed the spectroscopic discrimination of vesicles derived from different cell lines. To our knowledge, this work represents one of the first combined Raman and AFM-IR spectroscopic investigations of GPMVs, demonstrating the potential of multimodal vibrational spectroscopy for the characterization of cell-derived membrane systems. The presented methodology establishes a foundation for future spectroscopic studies of membrane-derived vesicles and their biochemical heterogeneity.