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 titanium by 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 by SEM. Titanium substrates were modified to create laser‐induced periodic surface structures (LIPSS) for visualization at the submicron scale. By 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; the viability of minimally irradiated cells was also 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 submicron surface features. The demonstrated method to track observed cell adhesion to submicron surface features by SEM has great implications for understanding cell migration on nanostructured surfaces as well as the exploration of simpler SEM preparation methods for cellular imaging.

Electron Microscopy Imaging Applications of Room Temperature Ionic Liquids in the Biological Field: A Review

“…featured a new treatment scheme that utilized a 5 % [EMI][BF 4 ] solution in McCoy's 5 A modified medium to image mammalian cells adhered to nanotopographic laser‐modified titanium substrates (Figure 5). [47] In Figure 5, low‐vacuum backscattered SEM imaging mode is used with the RTIL treatment, where cells appear dark in contrast to the bright titanium substrate. The work compared the influence of untreated titanium substrates (Figure 5A/B) and laser‐modified titanium (Figure 5C/D), where sub‐micron features induced from the laser‐modification appear as thin lines most clearly represented in Figure 5D [47] .…”

“… [47] In Figure 5, low‐vacuum backscattered SEM imaging mode is used with the RTIL treatment, where cells appear dark in contrast to the bright titanium substrate. The work compared the influence of untreated titanium substrates (Figure 5A/B) and laser‐modified titanium (Figure 5C/D), where sub‐micron features induced from the laser‐modification appear as thin lines most clearly represented in Figure 5D [47] . Prior to imaging, an optimal percentage of ionic liquid that would cause limited influence on cell viability while maintaining improved imaging in SEM was determined.…”

“…Prior to imaging, an optimal percentage of ionic liquid that would cause limited influence on cell viability while maintaining improved imaging in SEM was determined. Human osteoblast‐like Saos‐2 cells were imaged using SEM and subsequently evaluated using biochemical assays as well as fluorescence microscopy to confirm cell viability [47] . Cells from this specimen were viable after treatment with the RTIL‐media solution and after imaging; however, directly irradiated cells were killed due to electron beam damage [47] .…”

“…Human osteoblast‐like Saos‐2 cells were imaged using SEM and subsequently evaluated using biochemical assays as well as fluorescence microscopy to confirm cell viability [47] . Cells from this specimen were viable after treatment with the RTIL‐media solution and after imaging; however, directly irradiated cells were killed due to electron beam damage [47] . This demonstrates treatment applicability to facilitate live imaging of cells in near‐native environments while warning that cells are still highly vulnerable to the extreme environment required for EM.…”

“…Cells can be observed across the entire substrate of the unmodified sample (A/B) and in detail with respect to sub‐micron features from the laser‐modification treatment, which appear as fine lines on the metallic substrate (C/D). Image reproduced with permission from Bryan E. Lee et al [47] . (Copyright © 2020 Wiley‐VCH GmbH).…”