  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. CARS microscopy can detect
lipid membranes and droplet compartments in living cells and SHG microscopy
enables a strong imaging contrast for molecules with a non-centrosymmetric
ordered structure like collagen. 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
investigate the collagen production dynamic in living cell 3D cultures. Its
ability to image the cell morphology and the produced collagen distribution 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 addition the experiment shows
that the technique is a powerful tool for imaging of very thick sections (about
4 mm). The study conducted on mesenchymal stem cell in fibrin gel cultures
confirmed that differentiation stimulus is induced by the scaffold. The
monitoring of stem cell differentiation within a scaffold in a non-destructive
way will be an important advantage in regenerative medicine and tissue
engineering field.
