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.

中文翻译：

---

使用离子液体在SEM中同时可视化湿细胞和纳米结构生物材料的简便方法

这项工作提出了一种成功的方法，通过离子液体处理后，以低真空模式操作的扫描电子显微镜（SEM）对粘附在纳米结构生物材料上的哺乳动物细胞进行成像。用室温离子液体1-乙基-3-甲基咪唑四氟硼酸盐处理人成骨样Saos-2细胞，然后利用SEM在钛上成像。钛基材经过改性后可产生激光诱导的周期性表面结构（LIPSS），以亚微米级可视化。结合使用基于荧光的细胞代谢以及光学显微镜和SEM图像分析，可以确定经过多次辐照后，辐照细胞的形状和位置没有改变，而最小辐照细胞的活力没有改变。湿式成像条件与使用离子液体的快速简便操作方案相结合，使该技术在检查具有亚微米表面特征的生物材料上的细胞行为方面占据了一席之地。用SEM跟踪观察到的细胞对亚微米表面特征的粘附的方法对理解纳米结构表面上的细胞迁移以及探索用于细胞成像的更简单SEM制备方法具有重大意义。