Faithful Scanning Electron Microscopic (SEM) visualization of 3D printed alginate-based scaffolds

The morphological characterization of 3D printed hydrogel-based scaffolds is essential for monitoring their size, shape, surface texture and internal structure. Among other microscopic techniques, Scanning Electron Microscopy (SEM) is capable of visualizing nearly all kinds of materials at different length scales, with exceptional precision, if investigation under vacuum is possible. However, due to the high water content of hydrogel-based scaffolds and the connected volume change after drying, special preparation techniques are necessary to stabilize the 3D architecture when imaged by SEM. Here we present a straightforward cryo-SEM technique to visualize 3D printed hydrogel-based alginate scaffolds. By use of a homemade cryo-SEM holder and plunge-freezing in liquid ethane, scaffolds are visualized from the top and cross-sectional view at different magnifications. The proposed method is compared with SEM imaging in different modes (cyro-SEM, conventional SEM, ESEM) following other commonly used sample preparation techniques, such as plunging in liquid nitrogen, air-drying, freeze-drying and plunging in liquid ethane after graded dehydration. These approaches, except ESEM, lead to shrinkage, deformation, distortion or disintegration of the scaffolds and consequently give rise to artifacts in imaging. The presented results indicate that cryo-SEM after plunging in liquid ethane allows for the most faithful and time-efficient visualization of 3D printed alginate scaffolds.