Direct three-dimensional imaging for morphological analysis of electrospun fibers with laboratory-based Zernike X-ray phase-contrast computed tomography.

Electrospinning is a well-established and widely used method for the production of protein-based fibrous biomaterials. The visualization of the morphology and the characterization of sample features related to the three-dimensional (3D) structure, like the porosity and fibers thickness, is crucial for the design and fabrication of tailor-made and application-optimized materials. Here, we evaluated the benefits of using 3D X-ray imaging in a laboratory setup with a resolution in the sub-micrometer range for the characterization of electrospun gelatin fibrous mats. We used phase-contrast X-ray computed tomography at the nanoscale (nano-CT) for the evaluation of the time-course morphological changes of the mats induced by chemical cross-linking of the gelatin fibers. We present an image processing protocol that enables the segmentation of the fibers and quantification of the mats porosity, the analysis of the shape and size of the pores, and of the fibers thickness and orientation. We compared the results obtained from the processed nano-CT data with those obtained with the conventional methods used for the characterization of electrospun fibrous materials, and we discuss the advantages and limitations of each method when applied to gelatin electrospun samples. Our results reveal that the use of phase-contrast nano-CT provides quick additional and relevant information for the characterization of fibrous mats and, thus, provides beneficial insights for the design and fabrication of novel fibrous materials.