Ionic liquid treatment for efficient sample preparation of hydrated bone for scanning electron microscopy

This study presents a new protocol for preparing bone samples for scanning electron microscopy (SEM) using a room temperature ionic liquid (RTIL) treatment method. RTIL-based solutions can be adopted as an alternative to lengthy and laborious traditional means of preparation for SEM due to their unique low-vapour pressure and conductive properties. Applied to biological samples, RTILs can be used quickly and efficiently to observe hydrated, unfixed structures in typical SEM systems. This first-time feasibility study of the optimization of this protocol for bone was explored through various SEM modalities using two distinct ionic liquids, 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMI][BF₄]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMI][BF₄]), at varying concentrations of 5, 10, and 25 % v/v in aqueous solution through an addition-based method. Based on qualitative observations in the SEM, a 60-second solution addition treatment of 10 % v/v [BMI][BF₄] performed the best in imaging hydrated, unfixed bone samples, resulting in minimal charge buildup and no solution pooling on the surface. The treatment was applied effectively to a variety of bone samples, notably flat and polished, as well as highly topographical bone fracture surfaces of both healthy and osteoporotic human bone samples. In comparison to conventionally dehydrated bone, the RTIL treatment better preserved the natural bone structure, resulting in minimal microcracking in observed structures.