Compositional and structural characterization of dorsal root ganglion neurons and co-cultured Schwann cells by confocal Raman microspectral imaging

Abstract

Confocal Raman microspectral imaging (CRMI) is a versatile non-invasive technique that allows in vitro cell imaging without any chemical fixation, fluorescent markers or other contrast-enhancing chemicals. In this study, dorsal root ganglion (DRG) neuron and its affiliated Schwann cells (SCs) were co-cultured to unveil their underlying sub-cellular constitutional and structural nature. Both DRG neurons and SCs were derived from neonatal Sprague-Dawley rats and seeded on CaF₂ subtracts for spectral analysis. After acquiring Raman hyperspectral datasets, multivariate data analyses, including K-mean cluster analysis (KCA) and principal component analysis (PCA), were successively adopted to study the subcellular structural and compositional information of the measured cells. Univariate spectral analysis was adopted to emphasize the spatial distribution of subcellular constitutions based on the acquired spectral characteristics. Results have shown Raman spectral characteristics of DRG neurons (cell membrane, cytoplasm, organelles, nucleus) and its affiliated SCs (myelin, cell membrane, cytoplasm, nucleus), as well as information on the subcellular distribution pattern of major biochemical components (proteins, cytochrome c, nucleic acids, lipids, carbohydrates). This in vitro spectral-imaging work provides a proof of principle of an analytical method for future studies on the developmental mechanisms of DRG neurons and their molecular bases for the treatment of diseases of the peripheral nervous system.