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

Area of Science:

• Materials Science
• Biotechnology
• Microscopy

Background:

• Traditional bone sample preparation for scanning electron microscopy (SEM) is time-consuming and can damage delicate structures.
• Room temperature ionic liquids (RTILs) offer unique properties like low vapor pressure and conductivity, making them potential alternatives for biological sample preparation.
• Previous applications of RTILs in SEM have focused on various biological tissues, but their use for bone preparation requires specific optimization.

Purpose of the Study:

• To develop and optimize a novel protocol for preparing bone samples for SEM using room temperature ionic liquids (RTILs).
• To evaluate the efficacy of two distinct RTILs, [EMI][BF4] and [BMI][BF4], at various concentrations for bone sample preparation.
• To compare the RTIL-based method with conventional dehydration techniques in terms of structural preservation and imaging quality.

Main Methods:

• Exploration of a new RTIL-based protocol for bone sample preparation for SEM.
• Testing of two ionic liquids: 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMI][BF4]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMI][BF4]).
• Application of RTILs at concentrations of 5%, 10%, and 25% v/v in aqueous solution via an addition-based method, followed by SEM imaging.

Main Results:

• A 60-second treatment with 10% v/v [BMI][BF4] solution was identified as optimal for imaging hydrated, unfixed bone.
• This optimal RTIL treatment resulted in minimal charge buildup and no solution pooling on the bone surface.
• The RTIL method effectively imaged various bone types, including healthy and osteoporotic human bone, and preserved natural bone structure with reduced microcracking compared to dehydration.

Conclusions:

• Room temperature ionic liquid treatment presents a rapid and efficient alternative to traditional methods for preparing bone samples for SEM.
• The optimized [BMI][BF4] protocol significantly enhances the preservation of natural bone microstructure during SEM sample preparation.
• This novel RTIL approach facilitates the imaging of hydrated, unfixed bone, offering valuable insights into bone biology and pathology.