Bio-imaging with the helium-ion microscope: A review.

Scanning helium-ion microscopy (HIM) is an imaging technique with sub-nanometre resolution and is a powerful tool to resolve some of the tiniest structures in biology. In many aspects, the HIM resembles a field-emission scanning electron microscope (FE-SEM), but the use of helium ions rather than electrons provides several advantages, including higher surface sensitivity, larger depth of field, and a straightforward charge-compensating electron flood gun, which enables imaging of non-conductive samples, rendering HIM a promising high-resolution imaging technique for biological samples. Starting with studies focused on medical research, the last decade has seen some particularly spectacular high-resolution images in studies focused on plants, microbiology, virology, and geomicrobiology. However, HIM is not just an imaging technique. The ability to use the instrument for milling biological objects as small as viruses offers unique opportunities which are not possible with more conventional focused ion beams, such as gallium. Several pioneering technical developments, such as methods to couple secondary ion mass spectrometry (SIMS) or ionoluminescence with the HIM, also offer the possibility for new and exciting research on biological materials. In this review, we present a comprehensive overview of almost all currently published literature which has demonstrated the application of HIM for imaging of biological specimens. We also discuss some technical features of this unique type of instrument and highlight some of the new advances which will likely become more widely used in the years to come.

Antibacterial Efficiency of Surface-Immobilized Flavobacterium-Infecting Bacteriophage.

Control of bacterial diseases by bacteriophages (phages) is gaining more interest due to increasing antibiotic resistance. This has led to technologies to attach phages on surfaces to form a biomaterial that can functionally display phages that interact with bacteria, to carry out successful infection cycles. Such a material could be applied in many environments where the target pathogens are expected. Although this approach has been applied successfully in a few studies already, the basis of the antibacterial effect by the immobilized phages is unclear, and the interpretation of the results depends on the study. Here, we studied the phage attachment density, their detachment rate, and infectivity on five different surfaces: silicon, amine-treated silicon, gold, carboxylate-treated gold, and cross-linker-activated carboxylate-treated gold. The density of attached phages varied between the different surfaces and was the highest on the cross-linker-activated carboxylate-treated gold. To understand whether the antibacterial effect is caused by the attached or the detached phages, the strength of the immobilization was analyzed by performing 3-12 washing steps. The detachment rates differed between the materials, with the amine-treated silicon surface generating the highest release of phages and maintaining the highest infectivity, even after extensive washing. However, covalent cross-linking seemed to interfere with the infectivity. Our results suggest that the detachment of the phages from the surface is a possible mechanism for the antibacterial effect. Furthermore, we introduce a measure of the infectivity by comparing the bacterial growth reductions produced by the phage-treated materials to the effect caused by a known number of free phages, resulting in a unit "effective PFU/surface area", a comparable standard between different studies.

Bacteriophage imaging: past, present and future.

The visualization of viral particles only became possible after the advent of the electron microscope. The first bacteriophage images were published in 1940 and were soon followed by many other publications that helped to elucidate the structure of the particles and their interaction with the bacterial hosts. As sample preparation improved and new technologies were developed, phage imaging became important approach to morphologically classify these viruses and helped to understand its importance in the biosphere. In this review we discuss the main milestones in phage imaging, how it affected our knowledge on these viruses and recent developments in the field.

Beating Darwin-Bragg losses in lab-based ultrafast x-ray experiments.

The use of low temperature thermal detectors for avoiding Darwin-Bragg losses in lab-based ultrafast experiments has begun. An outline of the background of this new development is offered, showing the relevant history and initiative taken by this work.

Imaging Bacterial Colonies and Phage-Bacterium Interaction at Sub-Nanometer Resolution Using Helium-Ion Microscopy.

Imaging of microbial interactions has so far been based on well-established electron microscopy methods. This study presents a new way to study bacterial colonies and interactions between bacteria and their viruses, bacteriophages (phages), in situ on agar plates using helium ion microscopy (HIM). In biological imaging, HIM has advantages over traditional scanning electron microscopy with its sub-nanometer resolution, increased surface sensitivity, and the possibility to image nonconductive samples. Furthermore, by controlling the He beam dose or by using heavier Ne ions, the HIM instrument provides the possibility to mill out material in the samples, allowing for subsurface imaging and in situ sectioning. Here, the first HIM-images of bacterial colonies and phage-bacterium interactions are presented at different stages of the infection as they occur on an agar culture. The feasibility of neon and helium milling is also demonstrated to reveal the subsurface structures of bacterial colonies on agar substrate, and in some cases also structure inside individual bacteria after cross-sectioning. The study concludes that HIM offers great opportunities to advance the studies of microbial imaging, in particular in the area of interaction of viruses with cells.

Engineering thermal conductance using a two-dimensional phononic crystal.

Controlling thermal transport has become relevant in recent years. Traditionally, this control has been achieved by tuning the scattering of phonons by including various types of scattering centres in the material (nanoparticles, impurities, etc). Here we take another approach and demonstrate that one can also use coherent band structure effects to control phonon thermal conductance, with the help of periodically nanostructured phononic crystals. We perform the experiments at low temperatures below 1 K, which not only leads to negligible bulk phonon scattering, but also increases the wavelength of the dominant thermal phonons by more than two orders of magnitude compared to room temperature. Thus, phononic crystals with lattice constants ≥1 µm are shown to strongly reduce the thermal conduction. The observed effect is in quantitative agreement with the theoretical calculation presented, which accurately determined the ballistic thermal conductance in a phononic crystal device.