Effect of Ethanol Vapor Inhalation Treatment on Lethal Respiratory Viral Infection With Influenza A.

Ethanol (EtOH) effectively inactivates enveloped viruses in vitro, including influenza and severe acute respiratory syndrome coronavirus 2. Inhaled EtOH vapor may inhibit viral infection in mammalian respiratory tracts, but this has not yet been demonstrated. Here we report that unexpectedly low EtOH concentrations in solution, approximately 20% (vol/vol), rapidly inactivate influenza A virus (IAV) at mammalian body temperature and are not toxic to lung epithelial cells on apical exposure. Furthermore, brief exposure to 20% (vol/vol) EtOH decreases progeny virus production in IAV-infected cells. Using an EtOH vapor exposure system that is expected to expose murine respiratory tracts to 20% (vol/vol) EtOH solution by gas-liquid equilibrium, we demonstrate that brief EtOH vapor inhalation twice a day protects mice from lethal IAV respiratory infection by reducing viruses in the lungs without harmful side effects. Our data suggest that EtOH vapor inhalation may provide a versatile therapy against various respiratory viral infectious diseases.

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. In annular dark-field mode, the proper discrimination of electrons scattered by the specimen from those scattered by the background ice was found to be difficult due to the severe overlap of the scattered electrons. The resulting micrographs lacked clarity, and the ice-embedded phage particles could only be discerned after post-processing image adjustment. However, in annular bright-field mode, despite similar overlapping of the scattered electrons, it was possible to assess the morphology and intactness of the specimen in the embedding ice, suggesting that this mode may find utility in low-energy cryo-scanning transmission electron microscopy imaging methods.

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. These results confirm that the coherent portion of the direct beam defines the mean amplitude, and additionally show that the coherent portion corresponds to the conventional energy-filtered signal (with threshold 5 eV in this work). Finally, we present summary measurements from a selection of different materials, and compare the results against a simple electron scattering model. This study reinforces the claim that the mean amplitude is defined by the energy-filtered direct beam, and confirms that the contributions of elastic and inelastic scattering to the total mean free path are broadly in line with theoretical expectations for these different materials. These results in aggregate indicate that neither experimental collection angles nor enhanced sensitivity to low-loss phonon scattering affect the mean amplitude signal arising from off-axis holography reconstructions, nor the associated mean free path values which are derived from this mean amplitude.

Bragg holography of nano-crystals.

Crystal diffraction is a well-established technique for high-resolution structural analysis of material science and biological samples. However, the recovered structure is a result of averaging over all the unit cells in the crystal, which smears out the imperfections, atomic defects, or asymmetries and chiral properties of the individual molecules. We propose Bragg holography, where a nano-crystal is imaged at a defocus distance allowing separation of the diffracted beams, without turning them into peaks. The presence of a reference wave gives rise to a Bragg hologram, which can be reconstructed by conventional holographic reconstruction algorithms. The recovered complex-valued wavefront contains the complete information about the atomic distribution in the crystal, including defects. Bragg holography is demonstrated for gold nano-crystals, and its feasibility for biological nano-crystals is shown.

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. In this study, we present a solution to this issue by integrating cryo-EM capabilities into a commercial scanning electron microscope (SEM). Utilising the principle of low-energy in-line electron holography, our newly developed hybrid microscope permits low-to-moderate resolution imaging of vitreous ice-embedded biomolecules without the need for any form of sample staining or chemical fixation. Operating at 20 kV, the microscope takes advantage of the ease-of-use of SEM-based imaging and phase contrast imaging of low-energy electron holography. This study represents the first reported successful application of low-energy in-line electron holographic imaging to vitreous ice-embedded small biomolecules, the effectiveness of which is demonstrated here with three morphologically distinct specimens.

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. 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.

Development of a SEM-based low-energy in-line electron holography microscope for individual particle imaging.

A new SEM-based in-line electron holography microscope has been under development. The microscope utilizes conventional SEM and BF-STEM functionality to allow for rapid searching of the specimen of interest, seamless interchange between SEM, BF-STEM and holographic imaging modes, and makes use of coherent low-energy in-line electron holography to obtain low-dose, high-contrast images of light element materials. We report here an overview of the instrumentation and first experimental results on gold nano-particles and carbon nano-fibers for system performance tests. Reconstructed images obtained from the holographic imaging mode of the new microscope show substantial image contrast and resolution compared to those acquired by SEM and BF-STEM modes, demonstrating the feasibility of high-contrast imaging via low-energy in-line electron holography. The prospect of utilizing the new microscope to image purified biological specimens at the individual particle level is discussed and electron optical issues and challenges to further improve resolution and contrast are considered.

Water without windows: Evaluating the performance of open cell transmission electron microscopy under saturated water vapor conditions, and assessing its potential for microscopy of hydrated biological specimens.

We have performed open cell transmission electron microscopy experiments through pure water vapor in the saturation pressure regime (>0.6 kPa), in a modern microscope capable of sub-Å resolution. We have systematically studied achievable pressure levels, stability and gas purity, effective thickness of the water vapor column and associated electron scattering processes, and the effect of gas pressure on electron optical resolution and image contrast. For example, for 1.3 kPa pure water vapor and 300kV electrons, we report pressure stability of ± 20 Pa over tens of minutes, effective thickness of 0.57 inelastic mean free paths, lattice resolution of 0.14 nm on a reference Au specimen, and no significant degradation in contrast or stability of a biological specimen (M13 virus, with 6 nm body diameter). We have also done some brief experiments to confirm feasibility of loading specimens into an in situ water vapor ambient without exposure to intermediate desiccating conditions. Finally, we have also checked if water experiments had any discernible impact on the microscope performance, and report pertinent vacuum and electron optical data, for reference purposes.

Graphene specimen support technique for low voltage STEM imaging.

The scanning transmission electron microscopy (STEM) mode of today's field emission scanning electron microscopes enables sub-nanometer resolution imaging. Graphene is a single-atom thick, electrically conductive material, making it an excellent specimen support for the low voltage STEM imaging of nanometer-sized objects such as viruses. Here we present low voltage STEM images of bacteriophage T4 recorded on highly cleaned graphene films. The results show that ultrathin graphene support films markedly improve image signal at low accelerating voltages. Staining with a low atomic number methylamine vanadate stain combined with the graphene support film enables the clear visualization of the fine structure of the T4 tail by the low voltage STEM technique. Despite the advantages of graphene support films, difficulties are often encountered in placing hydrophilic biological samples on hydrophobic graphene electron microscopy grids. We employed a spin sedimentation sample loading method to overcome this problem.

Extreme ultraviolet free electron laser seeded with high-order harmonic of Ti:sapphire laser.

The 13th harmonic of a Ti:sapphire (Ti:S) laser in the plateau region was injected as a seeding source to a 250-MeV free-electron-laser (FEL) amplifier. When the amplification conditions were fulfilled, strong enhancement of the radiation intensity by a factor of 650 was observed. The random and uncontrollable spikes, which appeared in the spectra of the Self-Amplified Spontaneous Emission (SASE) based FEL radiation without the seeding source, were found to be suppressed drastically to form to a narrow-band, single peak profile at 61.2 nm. The properties of the seeded FEL radiation were well reproduced by numerical simulations. We discuss the future precept of the seeded FEL scheme to the shorter wavelength region.

Possibility of single biomolecule imaging with coherent amplification of weak scattering x-ray photons.

The number of photons produced by coherent x-ray scattering from a single biomolecule is very small because of its extremely small elastic-scattering cross section and low damage threshold. Even with a high x-ray flux of 3 x 10;{12} photons per 100-nm -diameter spot and an ultrashort pulse of 10 fs driven by a future x-ray free electron laser (x-ray FEL), it has been predicted that only a few 100 photons will be produced from the scattering of a single lysozyme molecule. In observations of scattered x rays on a detector, the transfer of energy from wave to matter is accompanied by the quantization of the photon energy. Unfortunately, x rays have a high photon energy of 12 keV at wavelengths of 1A , which is required for atomic resolution imaging. Therefore, the number of photoionization events is small, which limits the resolution of imaging of a single biomolecule. In this paper, I propose a method: instead of directly observing the photons scattered from the sample, we amplify the scattered waves by superimposing an intense coherent reference pump wave on it and record the resulting interference pattern on a planar x-ray detector. Using a nanosized gold particle as a reference pump wave source, we can collect 10;{4}-10;{5} photons in single shot imaging where the signal from a single biomolecule is amplified and recorded as two-dimensional diffraction intensity data. An iterative phase retrieval technique can be used to recover the phase information and reconstruct the image of the single biomolecule and the gold particle at the same time. In order to precisely reconstruct a faint image of the single biomolecule in Angstrom resolution, whose intensity is much lower than that of the bright gold particle, I propose a technique that combines iterative phase retrieval on the reference pump wave and the digital Fourier transform holography on the sample. By using a large number of holography data, the three-dimensional electron density map can be assembled.