Quantitative biorelevant profiling of material microstructure within 3D porous scaffolds via multiphoton fluorescence microscopy

This study presents a novel approach, based on fluorescence multiphoton microscopy (MPM), to image and quantitatively characterize the microstructure and cell-substrate interactions within microporous scaffold substrates fabricated from synthetic biodegradable polymers. Using fluorescently dyed scaffolds fabricated from poly(DTE carbonate)/poly(DTO carbonate) blends of varying porosity and complementary green fluorescent protein-engineered fibroblasts, we reconstructed the three-dimensional distribution of the microporous and macroporous regions in 3D scaffolds, as well as cellular morphological patterns. The porosity, pore size and distribution, strut size, pore interconnectivity, and orientation of both macroscale and microscale pores of 3D scaffolds were effectively quantified and validated using complementary imaging techniques. Compared to other scaffold characterizing techniques such as confocal imaging and scanning electron microscopy (SEM), MPM enables the acquisition of images from scaffold thicknesses greater than a hundred microns with high signal-to-noise ratio, reduced bulk photobleaching, and the elimination of the need for deconvolution. In our study, the morphology and cytoskeletal organization of cells within the scaffold interior could be tracked with high resolution within the limits of penetration of MPM. Thus, MPM affords a promising integrated platform for imaging cell-material interactions within the interior of polymeric biomaterials.