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In order to establish the resolving power of the HIM, a detailed analysis of the vitrified samples was performed. A remarkable feature of the HIM is that due to the high vacuum, it enables imaging of samples that are usually not accessible with other methods. The images of the low-vacuum, non-dehydrated samples show the complex nature of the biological surface in a wide field of view (Figure 5a-d). Some of the images reveal a complex, filamentous morphology of the cell walls, while others display a more crystalline, granular morphology of the surface (Figure 5c and d). In the case of the frozen, plunge-frozen and freeze-dried samples, the images clearly show that the surface is made up of different mineral particles, which are accessible via HIM due to the low-vacuum imaging conditions. Interestingly, the freeze-dried samples show a much smoother morphology of the surface in comparison to vitrified samples (Figure 5b). A potential explanation for this may be that during vitrification the EPS decreases the surface roughness, which is in contrast to the surface layer of EPS in freeze-dried samples, which might be the result of crystallization of the fragments of the EPS during the freezing process. The images of all of the samples also reveal cell information that is inaccessible using other techniques such as cryo-SEM or transmission electron microscopy. This includes information regarding the possible 3D organization of the cell wall and other intercellular components such as the cell-cell adhesion. The potential to examine the 3D organization of cell walls in a non-destructive way suggests that the method could be used in the future for in-vivo biological applications where the cell status can be monitored. Another advantage of HIM in comparison to FE-SEM is the fact that the images show more details on the roughness of the surface (Figure 6). This is very useful when studying the effects of smoothening agents (such as polyvinyl alcohol or polyethylene glycol (PEG) in the freeze-drying process), which can alter the surface roughness and potentially the chemistry of the cells16, which could be analyzed by X-ray, Raman, or infrared spectroscopy.
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