Low-energy scanning transmission electron microscopy applied to ice-embedded biological macromolecules.

In this report, we applied annular bright-field and annular dark-field low-energy (30 keV) scanning transmission electron microscopy imaging to a vitreous ice-embedded biological macromolecule, T4 phage, to investigate the applicability of these methods for morphological investigation and sample screening. Multiple camera lengths were examined to find the optimal acceptance angle for both modes. Image clarity differed substantially between the modes, with the presence of ice also strongly influencing the quality of acquired micrographs.

Interpretation of mean free path values derived from off-axis electron holography amplitude measurements.

In this work, we have explored the factors which govern mean free path values obtained from off-axis electron holography measurements. Firstly, we explore the topic from a theoretical perspective, and show that the mean amplitude reconstructed from off-axis holograms is due to the coherent portion of the direct, central object-transmitted beam only - it is not affected by the presence or absence of other scattered beams. Secondly, we present a detailed experimental study which compares mean free path values obtained from hologram sideband, centreband, EELS, and TEM measurements as a function of optical collection angle and energy-loss-filtering.

Low-energy in-line electron holographic imaging of vitreous ice-embedded small biomolecules using a modified scanning electron microscope.

Cryo-electron microscopy (cryo-EM) has become the method of choice in the field of structural biology, owing to its unique ability to deduce structures of vitreous ice-embedded, hydrated biomolecules over a wide range of structural resolutions. As cryo-transmission electron microscopes (cryo-TEM) become increasingly specialised for high, near-atomic resolution studies, operational complexity and associated costs serve as significant barriers to widespread usability and adoptability. To facilitate the expansion and accessibility of the cryo-EM method, an efficient, user-friendly means of imaging vitreous ice-embedded biomolecules has been called for.

Improved sample dispersion in cryo-EM using "perpetually-hydrated" graphene oxide flakes.

For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches.

Improved sample dispersion in cryo-EM using "perpetually-hydrated" graphene oxide flakes.

For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. Furthermore, we provide evidence that the presence of graphene oxide flakes in vitreous ice results in a significant reduction in electron beam-induced specimen decomposition.