Microspectroscopy of spectral biomarkers associated with human corneal stem cells

PURPOSE: Synchrotron-based radiation (SRS) Fourier-transform infrared (FTIR) microspectroscopy potentially provides novel biomarkers of the cell differentiation process. Because such imaging gives a “biochemical-cell fingerprint” through a cell-sized aperture, we set out to determine whether distinguishing chemical entities associated with putative stem cells (SCs), transit-amplifying (TA) cells, or terminally-differentiated (TD) cells could be identified in human corneal epithelium. METHODS: Desiccated cryosections (10 μm thick) of cornea on barium fluoride infrared transparent windows were interrogated using SRS FTIR microspectroscopy. Infrared analysis was performed through the acquisition of point spectra or image maps. RESULTS: Point spectra were subjected to principal component analysis (PCA) to identify distinguishing chemical entities. Spectral image maps to highlight SCs, TA cells, and TD cells of the cornea were then generated. Point spectrum analysis using PCA highlighted remarkable segregation between the three cell classes. Discriminating chemical entities were associated with several spectral differences over the DNA/RNA (1,425–900 cm(−1)) and protein/lipid (1,800–1480 cm(−1)) regions. Prominent biomarkers of SCs compared to TA cells and/or TD cells were 1,040 cm(−1), 1,080 cm(−1), 1,107 cm(−1), 1,225 cm(−1), 1,400 cm(−1), 1,525 cm(−1), 1,558 cm(−1), and 1,728 cm(−1). Chemical entities associated with DNA/RNA conformation (1,080 cm(−1) and 1,225 cm(−1)) were associated with SCs, whereas protein/lipid biochemicals (1,558 cm(−1) and 1,728 cm(−1)) most distinguished TA cells and TD cells. CONCLUSIONS: SRS FTIR microspectroscopy can be employed to identify differential spectral biomarkers of SCs, TA cells, and/or TD cells in human cornea. This nondestructive imaging technology is a novel approach to characterizing SCs in situ.