Molecular remodeling in neuronal apoptosis revealed by stimulated Raman scattering microscopy.

Apoptosis is characterized by extensive biochemical and structural remodeling, yet its subcellular molecular architecture remains incompletely understood. Stimulated Raman scattering (SRS) microscopy enables label-free, hyperspectral imaging of intracellular biochemical composition with submicron spatial resolution. However, prior SRS studies of apoptosis have reported inconsistent spectral signatures, possibly arising from uncontrolled sampling across heterogenous apoptotic stages. Here, we present stage-resolved hyperspectral SRS profiling of single neuronal HT22 cells to systematically delineate the structural and spectroscopic evolution of apoptosis. SRS imaging reveals distinct, stage-dependent morphological transformations accompanied by characteristic spectral signatures in the chemical fingerprint and high-wavenumber regions. Quantitative analyses further uncover dynamic redistribution of proteins, nucleic acids, and lipids between nuclear and cytoplasmic compartments throughout apoptotic progression. In particular, the protein-to-lipid ratio reflects stage-specific molecular reorganization. Our findings highlight previously unrecognized structural transitions and establish stage-resolved framework for molecular remodeling during apoptosis, providing new insights into the subcellular organization of apoptotic cell death.