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Fixed fibronectin-coated gold surfaces with and without adherent embryonic fibroblasts were probed via vibrational sum-frequency-generation (SFG) spectroscopy. The SFG spectra were compared to infrared reflection−absorption spectroscopy (IRRAS) data in the CH stretching region. Noticeable differences were observed in the IRRAS spectra of the samples, whereas SFG spectra of the same samples were largely similar. These results suggest that cells with their overall random distribution of CH groups do not contribute to the SFG spectra, resulting in similar spectral features related to the fibronectin coating regardless of whether cells are adhered to it. Furthermore, SFG spectra of cells adhered directly on gold were found to have features similar to those of cells adhered on fibronectin-covered gold. Additional experiments with living cells treated in vitro with the high-powered lasers used in these experiments did not result in any visible radiation damage to the cells. These results demonstrate the feasibility of using SFG spectroscopy as an experimental tool to characterize the extracellular matrix (ECM) layer adjacent to a gold substrate beneath a layer of cells and also suggest that this technique could be operated to examine the ECM in vitro.
