Ionic liquids are low-melting salts which are fluid at room temperature. They show a very low vapor pressure and are therefore persisting as liquids under the high vacuum conditions in an electron microscope.  They are electrical conductive as well. The ionic liquids tested are mixable with water, thus solutions can be easily diluted to suitable viscosities respectively concentrations.

In electron microscopy ionic liquids are used to coat surfaces and cavities to make samples conductive for SEM. This conductive treatment works on biological samples as well. In example, cell cultures or biofilms can be treated with ionic liquids. Preparation steps as heavy metal staining, dehydration and drying might be skipped. Cells get fixed for stopping life dynamics and structural stabilization and then directly treated with i.e. 10% Hitachi IL1000 before going into SEM without further coating.

Cells look adequate in SEM at lower magnification. At higher magnification structural collapse effects are visible. Occasionally charging phenomena can be observed. Additional block stain of the fixed sample with OsO₄ before ionic liquid treatment might attenuate collapse phenomena. Additional sputter coating after ionic liquid treatment prevents residual charging.

In an FIB/SEM cells treated with ionic liquids show after exposure to the ion beam no charging effects anymore; furthermore the backscatter electron signal is strongly enhanced. Cross sections of ionic liquid treated cells show a dense solid cytoplasm, which enables also consecutive sectioning for FIB/SEM-tomography.  While structural details are at best foreshadowed after ionic liquid treatment alone, substructures are visible after block staining with heavy metals before the treatment.

To find the relevant region of interest, correlation with light microscopic data is an option. Ionic liquid treatment doesn’t interfere with fluorescence detection in light microscopy. But also direct evaluation of characteristic topographic features of the sample surface can help to find the spot of interest for a FIB/SEM-tomogram.

Ionic liquid treatment makes biological samples vacuum resistant and conductive. The biofilm-substrate interface stays intact. These features are offered also by freeze-drying / critical-point-drying or thin-layer-plastification as well. Compared with other preparation techniques, ionic liquid treatment of biological specimen combines several advantages: preparation time is short, cell topography is clearly visible, cross sections show compact internal structures, fluorescence is preserved. But there are drawbacks as well: compared to freeze-drying or critical-point-drying, the surface is less well preserved; compared to thin-layer-plastification the internal structural is less clear exposed.

For FIB/SEM-studies, ionic liquid treatment is a compromise between critical-point-drying (excellent surface feature preservation, spongy aggregated structures inside the cells) and thin-layer-plastification (excellent internal structure preservation, poor topographic feature exposure). It is a quick SEM-preparation approach and the interface between cells and the substrate stays preserved.

Figures:

Fig.1. A - Cell culture on glass, fixed & treated with Hitachi IL 1000. Top view, SE-signal. B - Cells on posts, fixed, stained, IL-treated, sputtered, Pt-depo & FIB-cross section. Tilted view. BSE-signal.

Fig 2. Cells on glass, fixed & IL-treated. The middle cell only was beforehand irradiated with Ga-ions. Tilted view. C - SE-signal. D - BSE-signal.

Fig 3. FIB-cross sections of single cells on glass. Tilted views. The cells were fixed and stained, then dehydrated & critical point dried (E) or treated with Hitachi IL 1000 (F) or dehydrated & thin layer plastified in resin (G). Followed by sputter coating with platinum.

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EMC Abstracts - https://emc-proceedings.com/abstract/ionic-liquids-for-biological-sem-and-fibsem/