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Coherent anti‐Stokes Raman scattering (CARS) microscopy is used in conjunction with second harmonic generation (SHG) to follow the early stage of stem cell differentiation within a three‐dimensional (3D) scaffold. Formation of an extracellular matrix mainly composed of collagen is one of the first signs of human mesenchymal stem cell (hMSC) differentiation. This work shows that the multimodal CARS and SHG constitutes a powerful noninvasive, label‐free technique for monitoring collagen production in 3D cell cultures. Its simultaneous imaging of cell morphology and the distribution of the collagen produced by living cells during long‐term (4 weeks) experiment furnished important information about the cell‐scaffold interaction and the establishment of the extracellular matrix, while its very low collagen detection limit permitted mapping of even the small quantity produced in a few hours. This finding indicates that the multimodal CARS and SHG imaging can be proposed as a new way of monitoring the differentiation of stem cells by evaluating their production of collagen in both short and long‐term experiments. Further evidence is also provided of the efficacy of fibrin hydrogel as a scaffold autoinducing the differentiation stimulus in 3D human mesenchymal stem cell cultures. Copyright © 2012 John Wiley & Sons, Ltd.

CARS and SHG microscopy to follow collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin hydrogel 3D cultures