1 Center for Electron Nanoscopy, Technical University of Denmark2 Molecular Windows, Department of Micro- and Nanotechnology, Technical University of Denmark3 Department of Micro- and Nanotechnology, Technical University of Denmark4 DTU Admission Course, Technical University of Denmark5 Nanoprobes, Department of Micro- and Nanotechnology, Technical University of Denmark6 Fluidic Array Systems and Technology, Department of Micro- and Nanotechnology, Technical University of Denmark7 Department of Mechanical Engineering, Technical University of Denmark8 Center for Intelligent Drug Delivery and Sensing Using Microcontainers and Nanomechanics, Center, Technical University of Denmark9 Cell biology and virology Group, Biomedical Micro Systems Section, Department of Micro- and Nanotechnology, Technical University of Denmark10 University of Copenhagen
Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered.