Cryo dualbeam focused ion beam-scanning electron microscopy to evaluate the interface between cells and nanopatterned scaffolds

With the advance of nanotechnology in biomaterials science and tissue engineering, it is essential that new techniques become available to observe processes that take place at the direct interface between tissue and scaffold materials. Here, Cryo DualBeam focused ion beam–scanning electron microscopy (FIB-SEM) was used as a novel approach to observe the interactions between frozen hydrated cells and nanometric structures in high detail. Through a comparison of images acquired with transmission electron microscopy (TEM), conventional FIB-SEM operated at ambient temperature, and Cryo DualBeam FIB-SEM, the advantages and disadvantages of each technique were evaluated. Ultrastructural details of both (extra)cellular components and cell organelles were best observe with TEM. However, processing artifacts such as shrinkage of cells at the substrate interface were introduced in both TEM and conventional FIB-SEM. In addition, the cellular contrast in conventional FIB-SEM was low; consequently, cells were difficult to distinguish from the adjoining substrate. Cryo DualBeam FIB-SEM did preserve (extra)cellular details like the contour, cell membrane, and mineralized matrix. The three described techniques have proven to be complementary for the evaluation of processes that take place at the interface between tissue and substrat