Simple pretreatment of non-conductive small hydrous bio-samples with choline-type ionic liquid and membrane filter for microsample mounting

Choline-type ionic liquids (CILs), which have a molecular structure resembling that of the vitamin-like active substance choline, have high water solubility, high osmotic pressure, and high cell membrane permeability. Their physical properties make them a very useful pretreatment agent for scanning electron microscopy (SEM) observation of hydrous samples because they can replace water in the samples, causing them to retain their shapes. SEM images of pollen without dissolution of cell membranes, damage, or deformation were obtained by a simple visualization process using CILs. We also identified an easy pretreatment protocol for SEM observation of hydrous microsamples that uses a membrane filter as a sample stage to ensure correct sample placement. Bacteria were successfully observed by using this protocol.

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► Ionic liquids were used as a visualization reagent for SEM observation of hydrous samples.
► An IL with a molecular structure resembling that of choline has high water solubility and high cell membrane permeability.
► The choline-type IL provided a good SEM images of pollen without dissolution of cell membrane, damage or deformation.
► Polycarbonate membrane filter can be a good sample stage to ensure small hydrous sample by using the choline-type IL.