Characterizing Three-Dimensional Microstructure Of Collagen/Chitosan Scaffolds Using Multiphoton Microscope

In this work, we used multiphoton microscopic system for characterizing three-dimensional microstructure of collagen/chitosan polymeric scaffolds in a noninvasive fashion. Nonlinear optical signals including multiphoton autofluorescence (MAF) and second harmonic generation (SHG) derived from collagen/chitosan scaffolds were collected and analyzed. The three-dimensional porous microstructures of collagen/chitosan scaffolds were visualized by co-localized and evenly distributed MAF and SHG signals. The distribution of collagen and chitosan compositions within miscible collagen/chitosan blends cannot be either localized or differentiated simply using these nonlinear optical signals. However, the intensity of MAF signals in scaffolds was found to be markedly decreased in correlation to the supplementation of chitosan within blends, regardless of collagen/chitosan weight ratios. It therefore implied that the MAF-generating molecules within collagen being altered in miscible collagen/chitosan blends. And the SHG signals also decreased significantly in collagen/chitosan scaffolds with the supplementation of chitosan, regardless of different weight ratios. This finding supported the hypothesis regarding the miscibility of collagen/chitosan blends that triple helix structure of collagen, a proven SHG-generating microstructure, was altered in miscible collagen/chitosan blends. In conclusion, our work demonstrated that multiphoton imaging modality can be versatile for investigating three-dimensional microstructure of miscible polymeric scaffolds in a minimal invasive fashion, and may potentially be applicable in the field of tissue engineering.