Single-cell SR-FTIR spectroscopy reveals Aβ 25–35 -induced biomolecular alterations and cellular heterogeneity in HT22 cells

Alzheimer’s disease (AD) is a prevalent neurodegenerative disorder closely associated with neurotoxic aggregation of amyloid-β (Aβ) peptides, yet the subcellular and single-cell molecular mechanisms underlying Aβ-induced injury remain incompletely understood. In this work, HT22 cells were exposed to varying concentrations of Aβ25–35 to investigate the neurotoxic effects and cellular injury induced by the peptide. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy, a label-free technique combined with multivariate statistical analysis was used to characterize Aβ25–35-induced biomolecular alterations at the single-cell scale. Euclidean distances derived from single-cell spectra were further computed to quantitatively assess intercellular biomolecular heterogeneity. Spectral analysis revealed obvious changes in lipid structure and composition in all Aβ25–35-exposed cell groups. An increased β-sheet/α-helix ratio and a decreased nucleic acid-to-protein ratio were also identified, indicating alterations in protein conformation and nucleic acid content. Moreover, cells treated with varying Aβ25–35 exhibited distinct degrees of intercellular heterogeneity. These findings provide spectroscopic evidence for the neurotoxic effects of Aβ on HT22 cells, and demonstrate the single-cell SR-FTIR spectroscopy as a sensitive tool for probing biomolecular changes at cellular level.