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This information is obtained from secondary electron imaging. The effect of contamination and charging on our ability to extract such quantitative information will also be assessed. We will demonstrate that structural defects and stoichiometry modification can be controllably introduced in a few-layer molybdenum disulfide MoS 2 sample at a few-nanometer scale.

Consequently, localized tuning of the physical properties of MoS 2 can be realized. Fabrication of MoS 2 nanostructures with 7-nm dimensions and pristine crystal structure has also been achieved, and the effects of beam dose and profile on the modification will be clarified. This nanoscale modification technique is a generalized approach which can be applied to various 2D materials to produce a new range of 2D metamaterials.

The idea of using backscattered helium particles to access chemical information on the surface in a helium ion microscope came up right from the early days of this relatively young imaging technique. From the basic principles of backscattering spectrometry, ion solid interaction and particle detection it became clear rapidly that this attempt will suffer many difficulties in terms of technical realization and physical limitations.

This chapter is about describing those difficulties and working out different scenarios of how to apply backscattering spectrometry to the HIM anyways. Secondary Ion Mass Spectrometry SIMS is an extremely powerful technique for analysing surfaces, owing in particular to its excellent sensitivity , high dynamic range , very high mass resolution , and ability to differentiate between isotopes.

NIST Studies How New Helium Ion Microscope Measures Up | NIST

This chapter will discuss the feasibility of combining SIMS with Helium Ion Microscopy from a fundamental and instrumental point of view. After a sample has been excited by ion irradiation it has several ways to release the excess energy.

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One of them is photon emission. This process is called ionoluminescence IL. The IL signal contains information on the electronic structure of the sample. Furthermore, it can help to reveal the processes occurring in the sample under the influence of an ion beam. Analysis of IL is significantly complicated by the fact that ion beam not only induces light emission, but also modifies the luminescence properties of the material.

Several types of materials were investigated in HIM in terms of ionoluminescence: semiconductors, minerals, organic compounds. Analysis of the IL signal and its behavior allowed not only to identify the origin of the signal, but also to study the formation of ion-induced defects, their migration and interaction with each other. Such ionoluminescent patterns allowed the visualization and direct experimental measurements of the ion beam interaction volume. The performance of He-FIB milling and direct-write He-FIB induced deposition is compared to the performance achieved by other noble gas ions as well as the more conventionally used beams of electrons and Ga-ions.

Experimental results, simulations, and in-depth discussions of mechanisms highlight the peculiarities of each ion species with respect to nanostructuring issues like lateral sputter resolution, sputter rate, damage, amorphization, implantation, high-aspect ratio nanostructuring, deposition rate, chemical composition of deposits, and nanostructure shape fidelity. He-FIB is peculiar with respect to its small primary interaction volume and high secondary electron yield leading to excellent small milling and deposition features.

The other noble gas ions perform better than He with respect to unwanted ion implantation leading to swelling , higher sputter yields, or deposition of purer material from organometallic precursors. This chapter reviews focused He ion beam lithography, providing a detailed discussion on the ion beam-resist interaction mechanisms and latest experimental results in this field. In addition, impact of ion shot noise is examined, a comparison to He-ion beam milling is made, and future directions are mentioned.

The ability of gas field ion sources GFIS to produce controllable inert gas ion beams with atomic level precision opens up new applications in nanoscale direct-write material modification. Two areas where this has recently been demonstrated is focused helium ion beam production of high-transition temperature high- T C superconductor electronics and magnetic spin transport devices.

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The enabling advance in the case of superconducting electronics is the ability to use the GFIS to make features on the small length-scale of quantum mechanical tunnel barriers. Because the tunneling probability depends exponentially on distance, tunnel barriers must be less than a few nanometers wide, which is beyond the limits of other nanofabrication techniques such as electron beam lithography.

In magnetism, the GFIS has recently been used to generate chemical disordering and modify magnetic properties at the nanoscale. The strongest effect is observed in materials where ion-induced chemical disordering leads to increased saturation magnetization, enabling positive magnetic patterning. In this chapter, we review the latest results and progress in GFIS ion beam modification of high- T C superconductors and magnetic materials. Shane A.

Solid-state nanopores are an emerging technology for the detection and analysis of biomolecules at the single-molecule level. Additionally, the critical point dried samples show especially clean surfaces, whereas the surfaces of the minerals in plunge-frozen and freeze-dried samples appear to have a much smoother envelope in the case of the uncoated samples. We attribute this to a thin layer of extracellular polymeric substances EPS coating the whole aggregate. In contrast, we observed a relatively rough surface in the case of the platinum-coated samples see Supplementary Fig.

This indicates that sputter coating with platinum introduces an artificial surface roughness. Although the structures appeared much more flat in the uncoated sample, resolution was limited due to the necessity to use low-voltage imaging. The uncoated platelets in the HIM were for the first time revealed to be relatively flat with relatively sharp-tipped cellular attachments. HIM imaging has, for the first time, allowed the identification of this coating artifact because of the higher spatial resolution in combination with the capability of analyzing non-conductive, uncoated samples.

In addition, the existence of an EPS-envelope coating the minerals in plunge-frozen and freeze-dried samples could only be unambiguously shown in non-coated samples. Furthermore, sputter coating with platinum seemed only to affect the surface of the chemically fixed and critical point dried bacteria by creating a textured surface with network-like structures that created high secondary electron-signals Figure 4c.

We did not observe this effect when the cells were not coated with platinum Figure 4d. We also did not observe these structures on plunge-frozen and freeze-dried samples, both with and without the heavy metal coating see Supplementary Fig. Since dehydration in solvents such as ethanol a necessary step preceding critical point drying , can cause shrinkage in biological samples 34 , it is also possible that a similar effect could lead to a slightly textured cell surface that is potentially electrically insulating and thus, could favor localized deposition of platinum due to charge induced effects.

Finally the predator nematode, Pristionchus pacificus 35 served as an ultrastructural imaging challenge in the sense that the interior of the mouth cavity, including teeth morphology, had never been successfully imaged. A sister nematode, Parasitodiplogaster laevigata, a parasite of fig wasps has previously been shown via SEM imaging to have a protruding tooth structure and a visible Dorsal Esophogeal Gland Orifice DEGO located at the base of the protruding tooth However, in the initial HIM imaging of Pristionchus pacificus, it was revealed that a membranous sheath obscured the primary tooth structure Figure 5a.

This afforded an opportunity to employ another unique aspect of the HIM system — precision nano-machining using focused noble gas ions. By switching out the working gas of the HIM from helium to neon, it was possible to delicately remove the tip of the membranous sheath, while leaving the interior of the mouth cavity intact Figure 5c. This reveals the interior mouth structure as well as the primary tooth morphology and DEGO shape and size Figure 5b.

The very precise milling ability of the neon beam was effectively demonstrated by cutting the exterior of the nematode with a final dose of 0.

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This proved intense enough to cut through the outer skin of the worm without damaging any of the surrounding tissue. Despite being strong enough to penetrate the outer cuticle of the nematode, minimal ablation and thermal damage is visible on the surrounding area. Our results demonstrate both novel and exciting applications for helium ion microscopy in visualizing the surface ultrastructure of biological specimens at an unparalleled level of resolution and contrast. Moreover, the low sputtering rate of the helium ions beam for organic materials enables the repeated imaging of small and delicate surface features with no discernable beam damage.

Also, we show that the heavy metal coating as generally required for SEM introduces clearly resolvable structural artifacts and HIM is a superior method in clearly revealing surface features at high magnification. Finally, we couple a neon ion beam with the HIM setup allowing us to mill away surface membranes and provide us with unprecedented access to sub-surface structures.


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The viability of this approach centers on the sputter rates of neon being four times lower than that of gallium As such, the neon ion beam is ideally suited for the precision milling of very small and delicate biological structures. In addition, it causes far less discernable sample damage, or metal ion deposition, as is sometimes the case when using liquid metal ion sources such as gallium 37 , This higher resolution necessitates the development of new protocols for sample preparation, particularly to mitigate the possibility of fixation and drying artifacts.

Cryo-immobilization offers a proven and reliable alternative for the current fixation and dehydration approaches. We suggest that combining this cryo-based sample preparation paradigm with HIM will allow routine imaging with little or no artifacts. In conclusion we believe that this novel imaging method enabling the observation of completely unmodified structures on the nanometer scale will revolutionize our ability to interrogate both surface and sub-surface structures and provide deep scientific insights into the nanoscale architecture of biological specimens and soft materials.

After the drying stage, the samples were carefully removed and adhered to double-sided carbon tabs on aluminum stubs and stored in a desiccator. Wild type Arabidopsis were grown in soil to stage 6. All additional fill, heating, and venting steps were performed at medium speed as well. After drying, the samples were carefully removed and adhered to double-sided carbon tabs on aluminum stubs and stored in a desiccator.

Coated coverslips were then sterilized by UV light NuAire cell culture cabinet for 15 minutes on each side and stored until use. Cells were allowed to grow for 36—48 hours to ensure that the cells enter the exponential growth phase prior to fixation. Shorter periods of growth or over confluency yielded a much lower percentage of mitotic cells upon fixation. Cultures of the nitrate-reducing Acidovorax sp. Samples of the cell suspension were taken with syringes after 5 to 6 days of incubation when Fe II is completely oxidized This ensured bypassing the critical point when the pressure was released from 84 bars to atmospheric pressure.

To be able to compare samples treated after the standard sample preparation protocol of CPD with a more natural state of the cell-mineral aggregates, we decided to use a physical fixation rapid-freezing instead of chemical fixation, followed by freeze-drying under high vacuum. Therefore, droplets of the cell suspension were deposited onto formvar-coated mesh TEM-grids. The grids were stored in LN 2 until freeze-drying. Thermal conduction between the sample and the stage was ensured by a small droplet of liquid propane, which also prevented ice formation on the stage during sample transfer.

The progress of freeze-drying was followed visually. Pristionchus pacificus specimens strain identification: rlh24 were raised on agar plates coated with E. Worms then remained off food for 5 hours, providing sufficient time for P. This process is critical to produce a clean imaging sample.


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Bozzola, J. Electron microscopy: principles and techniques for biologists. Denk, W. PLoS Biol 2 , e, Stokes, D.


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