A Facile Method for Simultaneous Visualization of Wet Cells and Nanostructured Biomaterials in SEM using Ionic Liquids

Abstract This work presents a successful methodology to image mammalian cells adhered to nanostructured biomaterials using scanning electron microscopy (SEM) operating in low-vacuum mode following ionic liquid treatment. Human osteoblast-like Saos-2 cells were treated with a room-temperature ionic liquid, 1-Ethyl-3-methylimidazolium tetrafluoroborate, and subsequently imaged on titanium utilizing SEM. Titanium substrates were modified to create laser-induced periodic surface structures (LIPSS) for visualizing at the sub-micron scale. Using a combination of fluorescence-based cell metabolism along with light microscopy and SEM image analysis, the shape and location of irradiated cells were confirmed to be unchanged after multiple irradiation sessions while the viability of minimally irradiated cells was unaltered. The wet imaging conditions combined with a rapid facile protocol using ionic liquid allows this technique to fulfill a niche in examining cellular behavior on biomaterials with sub-micron surface features. The demonstrated method to track observed cell adhesion to sub-micron surface features with SEM has great implications for the understanding of cell migration on nanostructured surfaces as well as on the exploration of simpler SEM preparation methods for cellular imaging.