A facile pretreatment process for SEM: The use of room temperature ionic liquids (RTILs) provides an interesting method for SEM of biological specimens. We used a novel and concise method of pretreatment, excluding fixation or Au sputtering steps. Fine and smooth-textured SEM images of a wide variety of biological specimens treated in this way were observed without artefacts.

The cellular uptake of nanoparticles (NPs) was clearly observed by scanning electron microscopy (SEM) employing an ionic liquid for the first time. The samples did not show charge-up effect without staining, and high contrast images were achieved. Because of these unique properties of the ionic liquid, we could finely observe the cellular uptake of NPs compared with conventional SEM observation method. Moreover, the cells became transparent as the accelerating voltage increased, and the internalized NPs composed of heavy atoms were easily determined. This ionic liquid method provides a simple and short pretreatment procedure and thus is a promising technique for evaluating the cellular uptake of NPs and the intracellular fate of NPs. This method also gives a strategy to observe NPs which are unstable in the dry condition by choosing a suitable ionic liquid.

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