FIB-SEM imaging properties of Drosophila melanogaster tissues embedded in Lowicryl HM20.

Lowicryl resins enable processing of biological material for electron microscopy at the lowest temperatures compatible with resin embedding. When combined with high-pressure freezing and freeze-substitution, Lowicryl embedding supports preservation of fine structural details and fluorescent markers. Here, we analysed the applicability of Lowicryl HM20 embedding for focused ion beam (FIB) scanning electron microscopy (SEM) tomography of Drosophila melanogaster embryonic and larval model systems. We show that the freeze-substitution with per-mill concentrations of uranyl acetate provided sufficient contrast and an image quality of SEM imaging in the range of similar samples analysed by transmission electron microscopy (TEM). Preservation of genetically encoded fluorescent proteins allowed correlative localization of regions of interest (ROI) within the embedded tissue block. TEM on sections cut from the block face enabled evaluation of structural preservation to allow ROI ranking and thus targeted, time-efficient FIB-SEM tomography data collection. The versatility of Lowicryl embedding opens new perspectives for designing hybrid SEM-TEM workflows to comprehensively analyse biological structures. LAY DESCRIPTION: Focused ion beam scanning electron microscopy is becoming a widely used technique for the three-dimensional analysis of biological samples at fine structural details beyond levels feasible for light microscopy. To withstand the abrasion of material by the ion beam and the imaging by the scanning electron beam, biological samples have to be embedded into resins, most commonly these are very dense epoxy-based plastics. However, dense resins generate electron scattering which interferes with the signal from the biological specimen. Furthermore, to improve the imaging contrast, epoxy embedding requires chemical treatments with e.g. heavy metals, which deteriorate the ultrastructure of the biological specimen. In this study we explored the applicability of an electron lucent resin, Lowicryl HM 20, for focused ion beam scanning electron microscopy. The Lowicryl embedding workflow operates at milder chemical treatments and lower temperatures, thus preserving the sub-cellular and sub-organellar organization, as well as fluorescent markers visible by light microscopy. Here we show that focus ion beam scanning electron microscopy of Lowicryl-embedded fruit flies tissues provides reliable imaging revealing fine structural details. Our workflow benefited from use of transmission electron microscopy for the quality control of the ultrastructural preservation and fluorescent light microscopy for localization of regions of interest. The versatility of Lowicryl embedding opens up new perspectives for designing hybrid workflows combining fluorescent light, scanning, and transmission electron microscopy techniques to comprehensively analyze biological structures.

Alloy multilayers and ternary nanostructures by direct-write approach.

The fabrication of nanopatterned multilayers, as used in optical and magnetic applications, is usually achieved by two independent steps, which consist in the preparation of multilayer films and in the successive patterning by means of lithography and etching processes. Here we show that multilayer nanostructures can be fabricated by using focused electron beam induced deposition (FEBID), which allows the direct writing of nanostructures of any desired shape with nanoscale resolution. In particular, [Formula: see text] multilayers are prepared by the alternating deposition from the metal carbonyl precursors, [Formula: see text] and [Formula: see text], and neopentasilane, [Formula: see text]. The ability to fabricate nanopatterned multilayers by FEBID is of interest for the realization of hyperbolic metamaterials and related nanodevices. In a second experiment, we treated the multilayers by low-energy electron irradiation in order to induce atomic species intermixing with the purpose to obtain ternary nanostructured compounds. Transmission electron microscopy and electrical transport measurements indicate that in thick multilayers, (n = 12), the intermixing is only partial, taking place mainly in the upper part of the structures. However, for thin multilayers, (n = 2), the intermixing is such that a transformation into the L21 phase of the Co2FeSi Heusler compound takes place over the whole sample volume.

Direct writing of CoFe alloy nanostructures by focused electron beam induced deposition from a heteronuclear precursor.

Recently, focused electron beam-induced deposition has been employed to prepare functional magnetic nanostructures with potential in nanomagnetic logic and sensing applications by using homonuclear precursor gases like Fe(CO)5 or Co2(CO)8. Here we show that an extension towards the fabrication of bi-metallic compounds is possible by using a single-source heteronuclear precursor gas. We have grown CoFe alloy magnetic nanostructures from the HFeCo3(CO)12 metal carbonyl precursor. The compositional analysis indicates that the samples contain about 80 at% of metal and 10 at% of carbon and oxygen. Four-probe magnetotransport measurements are carried out on nanowires of various sizes down to a width of 50 nm, for which a room temperature resistivity of 43 µΩcm is found. Micro-Hall magnetometry reveals that 50 nm × 250 nm nanobars of the material are ferromagnetic up to the highest measured temperature of 250 K. Finally, the transmission electron microscopy (TEM) microstructural investigation shows that the deposits consist of a bcc Co-Fe phase mixed with a FeCo2 O4 spinel oxide phase with nanograins of about 5 nm diameter.

Synthesis of nanowires via helium and neon focused ion beam induced deposition with the gas field ion microscope.

The ion beam induced nanoscale synthesis of platinum nanowires using the trimethyl (methylcyclopentadienyl)platinum(IV) (MeCpPt(IV)Me3) precursor is investigated using helium and neon ion beams in the gas field ion microscope. The He(+) beam induced deposition resembles material deposited by electron beam induced deposition with very small platinum nanocrystallites suspended in a carbonaceous matrix. The He(+) deposited material composition was estimated to be 16% Pt in a matrix of amorphous carbon with a large room-temperature resistivity (∼3.5 × 10(4)-2.2 × 10(5) µΩ cm) and temperature-dependent transport behavior consistent with a granular material in the weak intergrain tunnel coupling regime. The Ne(+) deposited material has comparable composition (17%), however a much lower room-temperature resistivity (∼600-3.0 × 10(3) µΩ cm) and temperature-dependent electrical behavior representative of strong intergrain coupling. The Ne(+) deposited nanostructure has larger platinum nanoparticles and is rationalized via Monte Carlo ion-solid simulations which show that the neon energy density deposited during growth is much larger due to the smaller ion range and is dominated by nuclear stopping relative to helium which has a larger range and is dominated by electronic stopping.

Room temperature L10 phase transformation in binary CoPt nanostructures prepared by focused-electron-beam-induced deposition.

CoPt-C binary alloys have been fabricated by focused-electron-beam-induced deposition by the simultaneous use of Co2(CO)8 and (CH3)3CH3C5H4Pt as precursor gases. The alloys are made of CoPt nanoparticles embedded in a carbonaceous matrix. TEM investigations show that as-grown samples are in an amorphous phase. By means of a room temperature low-energy electron irradiation treatment the CoPt nanoparticles transform into face-centered tetragonal L10 nanocrystallites. In parallel, the system undergoes a transition from a superparamagnetic to a ferromagnetic state at room temperature. By variation of the post-growth irradiation dose the electrical and magneto-transport properties of the alloy can be continuously tuned.

Artificial granularity in two-dimensional arrays of nanodots fabricated by focused-electron-beam-induced deposition.

We have prepared 2D arrays of nanodots embedded in an insulating matrix by means of focused-electron-beam-induced deposition using the W(CO)(6) precursor. By varying the deposition parameters, i.e. the electron beam current and energy and the raster constant, we obtain an artificial granular material with tunable electrical properties. The analysis of the temperature dependence of the conductivity and of the current-voltage characteristic suggests that the transport mechanism is governed by electron tunneling between artificial grains. In order to understand the nature of the granularity and thus the microstructural origin of the electronic transport behavior, we perform TEM and micro-Raman investigations. Independent of the deposition parameters, TEM measurements show that the dots are constituted of amorphous tungsten carbide clusters embedded in an amorphous carbonaceous matrix. Micro-Raman spectra show two peaks, around 690 and 860 cm(-1) associated with the W-C stretching modes. Higher frequency peaks give information on the composition of the matrix. In particular, we measure a peak at about 1290 cm(-1), which is associated with sp(3) carbon bonds. Furthermore we detect the so-called D and G peaks, at about 1350 and 1560 cm(-1), associated with the vibration modes of the sp(2) carbon bonds. The analysis of the position of the peaks and of their relative intensity suggests that the composition of the matrix is between nanocrystalline graphite and amorphous carbon.

Ultra-fast vortex motion in a direct-write Nb-C superconductor.

The ultra-fast dynamics of superconducting vortices harbors rich physics generic to nonequilibrium collective systems. The phenomenon of flux-flow instability (FFI), however, prevents its exploration and sets practical limits for the use of vortices in various applications. To suppress the FFI, a superconductor should exhibit a rarely achieved combination of properties: weak volume pinning, close-to-depairing critical current, and fast heat removal from heated electrons. Here, we demonstrate experimentally ultra-fast vortex motion at velocities of 10-15 km s-1 in a directly written Nb-C superconductor with a close-to-perfect edge barrier. The spatial evolution of the FFI is described using the edge-controlled FFI model, implying a chain of FFI nucleation points along the sample edge and their development into self-organized Josephson-like junctions (vortex rivers). In addition, our results offer insights into the applicability of widely used FFI models and suggest Nb-C to be a good candidate material for fast single-photon detectors.

The transient electrical conductivity of W-based electron-beam-induced deposits during growth, irradiation and exposure to air.

W-based granular metals have been prepared by electron-beam-induced deposition from the tungsten hexacarbonyl W(CO)(6) precursor. In situ electrical conductivity measurements have been performed to monitor the growth process and to investigate the behavior of the deposit under electron beam post-irradiation and by exposure to air. During the first part of the growth process, the electrical conductivity grows nonlinearly, independent of the electron beam parameters. This behavior is interpreted as the result of the increase of the W-particle's diameter. Once the growth process is terminated, the electrical conductivity decreases with the logarithm of time, sigma approximately ln(t). Temperature-dependent conductivity measurements of the deposits reveal that the electrical transport takes place by means of electron tunneling either between W-metal grains or between grains and trap sites in the matrix. After venting the electron microscope the electrical conductivity of the deposits shows a degradation behavior, which depends on the composition. Electron post-irradiation increases the electrical conductivity of the deposits.

Magnetoresistance of granular Pt-C nanostructures close to the metal-insulator transition.

We investigate the electrical and magneto-transport properties of Pt-C granular metals prepared by focused electron beam induced deposition. In particular, we consider samples close to the metal-insulator transition obtained from as-grown deposits by means of a low-energy electron irradiation treatment. The temperature dependence of the conductivity shows a σ ∼lnT behavior, with a transition to σ ∼ √T at low temperature, as expected for systems in the strong coupling tunneling regime. The magnetoresistance is positive and is described within the wavefunction shrinkage model, normally used for disordered systems in the weak coupling regime. In order to fit the experimental data, spin-dependent tunneling has to be taken into account. In the discussion we attribute the origin of the spin-dependency to the confinement effects of Pt nano-grains embedded in the carbon matrix.