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Collecting information at the interface between living cells and artificial substrates is exceedingly difficult. The extracellular matrix (ECM) mediates all cell-substrate interactions, and its ordered, fibrillar constituents are organized with nanometer precision. The proceedings at this interface are highly dynamic and delicate. In order to understand factors governing biocompatibility or its counterpart antifouling, it is necessary to probe this interface without disrupting labels or fixation and with sufficient temporal resolution. Here the authors combine nonlinear optical spectroscopy (sum-frequency-generation) and microscopy (second-harmonic-generation), fluorescence microscopy, and quartz crystal microgravimetry with dissipation monitoring in a strategy to elucidate molecular ordering processes in the ECM of living cells. Artificially (fibronectin and collagen I) and naturally ordered ECM fibrils (zebrafish, Danio rerio) were subjected to nonlinear optical analysis and were found to be clearly distinguishable from the background signals of diffusive proteins in the ECM. The initial steps of fibril deposition and ordering were observed in vitro as early as 1 h after cell seeding. The ability to follow the first steps of cell-substrate interactions in spite of the low amount of material present at this interface is expected to prove useful for the assessment of biomedical and environmental interfaces.

In vitro observation of dynamic ordering processes in the extracellular matrix of living, adherent cells