Stable protein expression interference of key components of transforming growth factor (TGF)-β pathway in fibroblasts was achieved by transfection with a shRNA lentivirus. Two-photon microscopy, combined with an image informatics framework that suitable for 3D cellmatrix system, was used to study the TGF-β signaling in fibroblasts inside porous collagen scaffolds. It is concluded that, compared with TGF-β3, TGF-β1 plays a more important role in mediating myofibroblast differentiation, and that SMAD1 and SMAD3 play key roles in TGF-β1 mediated myofibroblast differentiation but in different ways. These conclusions were consistent with the results obtained by using biochemical or genetic methods.