Evaluation of Maltose-based Mixtures With Lanthanoid Acetates as Electron Staining Reagents for Transmission Electron Microscopy of Extracellular Vesicles and Liposomes

Abstract Extracellular vesicles (EVs), including exosomes, have attracted interest as vehicles for intercellular communication. Recognition of EVs as bio-derived nanocolloids allows for advanced fractionation and preparation methods that preserve their integrity. Direct observation of EVs is critical, and negative staining with heavy metal salts is a standard technique in biological transmission electron microscopy for routine morphological analysis and experimental optimization. However, customizations are often required for specific objectives. Uranyl acetate, widely used for its excellent electron scattering properties, allows clear visualization of ultrafine structures but is limited by strict global regulations and its harsh effects on biological membranes. To overcome these challenges, the author investigated lanthanoid series heavy metal salts as alternatives. Among various maltose-based mixtures, a combination of lanthanum and samarium acetates proved effective for observing EV morphology. In addition, replacing maltose with trehalose, which is known to increase water retention, resulted in improved staining characteristics with lanthanum and samarium acetates.