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

Coherent anti-Stokes Raman scattering (CARS) microscopy is combined with second harmonic generation (SHG) technique in order to follow the early stage of stem cell differentiation within a 3D scaffold. One of the first evidence of hMSCs differentiation is the formation of an extracellular matrix (ECM) where the collagen protein is its main component. This work demonstrated the multimodal CARS and SHG microscopy as a powerful non-invasive label free technique to follow the collagen production in living cell 3D cultures. Its ability to image the cell morphology and the produced collagen distribution on the same sample at the same time, on a long term (4 weeks) experiment allowed to obtain important information about the cell-scaffold interaction and the ECM production. The very low limit reached in detecting collagen has permitted to map even the small amount of collagen produced by the cells in few hours of culture. This demonstrates multimodal CARS and SHG microscopy as a novel method to follow cells collagen production and cells differentiation process in both short and long term experiments. In addition the experiment shows that the technique is a powerful tool for imaging of very thick sections (about 4 mm) with several advantages in its applications. As collagen production is considered a biomarker for ECM production and also a signal of initial stem cells differentiation, the study conducted on