gms | German Medical Science

Objective: The analysis of single cells with regards to their differentiation status and cell type of origin are frequent investigations in neurobiology and oncology. Currently, the analysis of cell type and cellular differentiation is only possible by altering the cells, mostly by fixation and consecutive staining. Spectroscopic optical imaging allows to analyze the molecular signature of solid materials, tissues and single cells. This project aims to develop a touchless method to identify of the cell type under investigation, in particular the determination and analyses of cell fate during the differentiation of neural stem cells.

Methods: Primary murine cerebellar embryonic neural precursor cells were grown as neurospheres. Differentiation was induced by removal of growth factor containing media and the addition of horse serum. Two different spectroscopic methods were employed to analyze a cellular monolayer. Infrared spectroscopy and Raman spectroscopy were used to generate spectrographic maps of plated cells. For comparison of the spectroscopic results with the current gold standard represented by immunohistochemistry, cells were freeze dried and consecutively analyzed by spectroscopy, then by immunohistochemistry. The following antibodies against differentiation markers were used to determine neural cell fate: nestin, GFAP, β3-tubulin, NeuN, HucC, Galc, and CNPase.

Results: Spectroscopic maps of native specimens of patients with human gliomas were collected ex vivo by infrared (IR) spectroscopy. Variations of cellular composition within each spectroscopic map were demonstrated by cluster analysis and were compared to immunohistochemical findings. For Raman spectroscopy, spectral differences in the data set were identified by principal component analysis (PCA) and matched to specific neural cell types.

Conclusions: Differentiated cells were successfully distinguished by infrared spectroscopy. These results are currently adapted to Raman spectroscopy, which has promising potential as an analytical tool for biomedical applications because it can probe the chemical composition and molecular structure of single cells and because its ability to probe samples under in vivo conditions. These tools should be of advantage in regenerative medicine and for monitoring of phenotypic alterations of tumor cells during tumor progression and therapy.