Background: Disruption of the microstructure in corneal stroma can lead to the loss of transparency. The lack of a characterization method for the microstructure prevents such scaffolds to be implemented in tissue transplantation. The non-invasive, three-dimensional (3D) rendering multiphoton microscopy (MPM) poses the potential to solve this problem.

Methods: MPM images and data analyses were performed with three kinds of samples with known and different quality. Isosurfaces (ISOs) were constructed for the evaluation of void volume and collagen distribution.

Results: The differences in the microstructures of these samples were revealed with clear indications and links to their behaviours in rehydration and possible transparency. According to this analysis, the scaffold with the highest void space ratio amongst the three presented the highest successful rates to be thoroughly rehydrated.

Conclusions: Such a method can be developed for assessing the quality of tissue engineered corneas, or donated corneas, and be useful as a powerful research tool in cornea related research.