In Situ Ni²⁺ Stain for Liposome Imaging by Liquid-Cell Transmission Electron Microscopy

Solvated soft matter, both biological and synthetic, can now be imaged in liquids using liquid-cell transmission electron microscopy (LCTEM). However, such systems are usually composed solely of organic molecules (low Z elements) producing low contrast in TEM, especially within thick liquid films. We aimed to visualize liposomes by LCTEM rather than requiring cryogenic TEM (cryoTEM). This is achieved here by imaging in the presence of aqueous metal salt solutions. The increase in scattering cross-section by the cation gives a staining effect that develops in situ, which could be captured by real space TEM and verified by in situ energy dispersive x-ray spectroscopy (EDS). We identified beam-induced staining as a time-dependent process that enhances contrast to otherwise low contrast materials. We describe the development of this imaging method and identify conditions leading to exceptionally low electron doses for morphology visualization of unilamellar vesicles before beam-induced damage propagates.