As reviewed above, it is clear that no single technique can fulfill all the high-resolution imaging needs in tissue engineering. The spatial imaging scale and the contrast mechanism of the technique determine, but also limit, the information it can provide and thus inform the range of its applicability. To overcome this limitation, multimodality imaging, the combination of generally two or three imaging techniques, has been employed to achieve a more robust assessment of a sample with multidimensional information.
An integrated OCM and 2-PFM system was built by Tan et al. to simultaneously provide optical scattering and fluorescence imaging from the same sample region (Tan et al., 2007; Graf and Boppart, 2010). Specifically, the 2-PFM can image individual cells, and the OCM allows structural visualization of the substrates. This multimodal approach has been applied to investigate cellular responses to mechanical stimuli in 3-D tissue constructs through the imaging of fibroblasts cultured in 3-D PDMS substrates and polymer scaffolds, (Tan et al., 2007). The combination of OCT and CFM has been used for longitudinal visualization of the cell dynamics in chitosan scaffold culture (Graf and Boppart, 2010). Dunkers et al. have reported a collinear OCM and CFM instrument for simultaneous scattering and fluorescence imaging deep inside a PCL scaffold with fetal chick osteoblasts (Dunkers et al., 2003). Complementary information provided by OCT and SHGM has been applied for time-dependent monitoring of the wound-healing process in a skin- equivalent raft tissue (Yeh et al., 2004). In this approach, OCT imaging was used to delineate the injured region and collagen microstructure organization, while fibroblast cell migration in response to injury was visualized by SHGM (Yeh et al., 2004). In addition, since both rely on two-photon excitation, 2-PFM and SHGM are often combined to image collagen-related tissue constructs and cell behavior in tissue engineering (Villa et al., 2013; Filova et al., 2010; Schenke-Layland et al., 2003; Cortiella et al., 2010). As an example, the extracellularly distributed collagen in a PCL scaffold was visualized, and the volume of collagen was quantified using combined 2-PFM and SHGM imaging (Filova et al., 2010).