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

Abstract

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.

Hybrid nanocapsules for in situ TEM imaging of gas evolution reactions in confined liquids

“…30 In a similar manner, sub-1 µm liposomes could be used as electron-transparent vessels for controlling and imaging nanoscale reactions in TEM liquid cells. Towards the latter objective, empty liposomes 50 to 300 nm in diameter, [39][40][41] along with other soft colloids like polymeric micelles 42,43 and vesicles, 44 have been imaged with liquid cell TEM. Due to potential electron beam damage, however, it remains a challenge to utilize these soft colloids as nanoreactors for hosting reactions for high resolution in situ TEM imaging.…”

“…Generally, bubbles are not desired during in situ experiments and their formation inside nanocapsules can be avoided by imaging at low dose rates. [39][40][41] However, here we opted to study the evolution of nanobubbles as a model reaction, because there are a number of advantages this system can offer. For instance, the high contrast of the nanobubbles facilitates automated tracking ( Figure S5), their size indicates the extent of radiolysis reactions, and their expansion allows us to test the mechanical stability of the nanocapsules.…”

“…65 In addition to formation of bubbles, the reactive by-products of water radiolysis 62 can attack organic functional groups in liposomes. 41 For instance, hydroxyl radicals (OH•) cause hydrogen abstraction, oxidative degradation and formation of radical anions on lipids that can facilitate complexation with free metal cations in the solution. 41 Hence, lipids could sequester ions meant for chemical reactions and could subsequently serve as nucleation sites.…”

“…41 For instance, hydroxyl radicals (OH•) cause hydrogen abstraction, oxidative degradation and formation of radical anions on lipids that can facilitate complexation with free metal cations in the solution. 41 Hence, lipids could sequester ions meant for chemical reactions and could subsequently serve as nucleation sites. Such beam effects can be mitigated by careful management of dose rates and beam exposureonly using higher doses when minimizing noise is critical.…”

“…These liquid cell TEM experiments would be analogous to liposome-mediated reactions imaged by optical microscopy, [30][31][32]38 but with orders of magnitude higher spatial resolution. While liposome fusion does not require high electron dose rates and can be initiated at low doses, 40,41 it does require two nanocapsules to be in close proximity. Deterministic approaches for pairing nanocapsules loaded with complementary reactants can be implemented by functionalizing liposomes with lipids of opposite charge, 66 by creating nested structures of liposomes in liposomes, 35,96 or by hydrodynamically trapping 97 liposomes in features patterned on the window membranes.…”