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Cell survival and infiltration into tissue engineering scaffolds affects both the rate at which cells proliferate on the scaffold and the ultimate functionality of the scaffold. In order to assess cell distribution and growth in degradable scaffolds, we have developed a combination of methods that results in a 3D image that can be used to assess cell viability and distribution within PLGA scaffolds. These methods involve first labeling live cells with specific fluorescence dyes, sectioning of the scaffold construct, compilation of fluorescence images, deconvolution, and reassembly into a 3D image. Our results have shown cell seeding by orbital shaking is the best method to seed cells while causing minimal cell death due to stress and/or media deprivation while allowing cell distribution throughout the entire scaffold. In addition, analysis of the compiled images shows that most cells reside on the surface and in the porous area near the scaffold surface after static seeding.