Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG.

Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition.

High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa.

We examined the rat and mouse epididymis using helium ion microscopy (HIM), a novel imaging technology that uses a scanning beam of He(+) ions to produce nanometer resolution images of uncoated biological samples. Various tissue fixation, sectioning and dehydration methods were evaluated for their ability to preserve tissue architecture. The cauda epididymidis was luminally perfused in vivo to remove most spermatozoa and the apical surface of the epithelial lining was exposed. Fixed epididymis samples were then subjected to critical point drying (CPD) and HIM. Apical stereocilia in principal cells and smaller apical membrane extensions in clear cells were clearly distinguishable in both rat and mouse epididymis using this technology. After perfusion with an activating solution containing CPT-cAMP, a permeant analog of cAMP, clear cells exhibited an increase in the number and size of membrane ruffles or microplicae. In contrast, principal cells did not exhibit detectable structural modifications. High-resolution HIM imaging clearly showed the ultrastructure of residual sperm cells, including the presence of concentric rings on the midpiece, and of cytoplasmic droplets in some spermatozoa. Close epithelium-sperm interactions were also detected. We found a number of sperm cells whose heads were anchored within the epididymal epithelium. In certain cases, the surface of the sperm cytoplasmic droplet was covered with vesicle-like structures whose size is consistent with that of epididymosomes. In conclusion, we describe here the first application of HIM technology to the study of the structure and morphology of the rodent epididymis. HIM technology represents a major imaging breakthrough that can be successfully applied to study the epididymis and spermatozoa, with the goal of advancing our understanding of their structure and function.

Switching modes in easy and hard axis magnetic reversal in a self-assembled antidot array.

We study the reversal mechanisms in a self-assembled, hexagonally ordered Fe antidot array with a period of 200 nm and an antidot diameter of 100 nm which was prepared by polystyrene nanosphere lithography. Direction-dependent information in such a self-assembled sample is obtained by measuring the anisotropic magnetoresistance (AMR) through constrictions processed by focused ion beam milling in nearest neighbor and next nearest neighbor directions. We show that such an originally integral method can be used to investigate the strong in-plane anisotropy introduced by the antidot lattice. The easy and hard axis reversal mechanisms and corresponding AMR signals are modeled by micromagnetic simulations. Additional in-field magnetic force microscopy studies allow the correlation of microscopic switching to features in the integral AMR. We find that the easy axis of magnetization is connected to a distinct periodic magnetic domain pattern, which can be observed during the whole magnetization reversal. While this process is driven by nucleation and propagation of reversed domains, the hard axis reversal is characterized by a (stepwise) rotation of the magnetization via the antidot lattice' easy axes.

Improved focused ion beam target preparation of (S)TEM specimen--a method for obtaining ultrathin lamellae.

Specimen quality is vital to (scanning) transmission electron microscopy (TEM) investigations. In particular, thin specimens are required to obtain excellent high-resolution TEM images. Conventional focused ion beam (FIB) preparation methods cannot be employed to reliably create high quality specimens much thinner than 20 nm. We have developed a method for in situ target preparation of ultrathin TEM lamellae by FIB milling. With this method we are able to routinely obtain large area lamellae with coplanar faces, thinner than 10 nm. The resulting specimens are suitable for low kV TEM as well as scanning TEM. We have demonstrated atomic resolution by Cs-corrected high-resolution TEM at 20 kV on a FIB milled Si specimen only 4 nm thick; its amorphous layer measuring less than 1 nm in total.

Periphery-substituted [4+6] salicylbisimine cage compounds with exceptionally high surface areas: influence of the molecular structure on nitrogen sorption properties.

The synthesis of various periphery-substituted shape-persistent cage compounds by twelve-fold condensation reactions of four triptycene triamines and six salicyldialdehydes is described, where the substituents systematically vary in bulkiness. The resulting cage compounds were studied as permanent porous material by nitrogen sorption measurements. When the material is amorphous, the steric demand of the cages exterior does not strongly influence the gas uptake, resulting in BET surface areas of approximately 700 m(2) g(-1) for all cage compounds 3 c-e, independently of the substituents bulkiness. In the crystalline state, materials of the same compounds show a strong interconnection between steric demand of the peripheral substituent and the resulting BET surface area. With increasing bulkiness, the overall BET surface area decreases, for example 1291 m(2) g(-1) (for cage compound 3 c with methyl substituents), 309 m(2) g(-1) (for cage compound 3 d with 2-(2-ethyl-pentyl) substituents) and 22 m(2) g(-1) (for cage compound 3 e with trityl substituents). Furthermore, we found that two different crystalline polymorphs of the cage compound 3 a (with tert-butyl substituents) differ also in nitrogen sorption, resulting in a BET surface area of 1377 m(2) g(-1), when synthesized from THF and 2071 m(2) g(-1), when recrystallized from DMSO.

Microwave reflection measurement of critical currents in a nanotube Josephson transistor with a resistive environment.

A scheme for measuring small intrinsic critical currents I(c) in nanoscale devices is described. Changes in Josephson inductance L(J) are converted to frequency variations that are recorded via microwave reflection measurements at 700-800 MHz. The critical current is determined from the frequency shift of the reflection magnitude at zero phase bias assuming a sinusoidal current-phase relation. The method is used to study a multiwalled carbon nanotube transistor with Pd/Nb contacts inside a resistive on-chip environment. We observe gate-tunable critical currents up to I(c) ∼ 8 nA corresponding to L(J) > 40 nH. The method presented is also applicable to devices shunted by closed superconducting loops.

Strong gate coupling of high-Q nanomechanical resonators.

The detection of mechanical vibrations near the quantum limit is a formidable challenge since the displacement becomes vanishingly small when the number of phonon quanta tends toward zero. An interesting setup for on-chip nanomechanical resonators is that of coupling them to electrical microwave cavities for detection and manipulation. Here we show how to achieve a large cavity coupling energy of up to (2π) 1 MHz/nm for metallic beam resonators at tens of megahertz. We used focused ion beam (FIB) cutting to produce uniform slits down to 10 nm, separating patterned resonators from their gate electrodes, in suspended aluminum films. We measured the thermomechanical vibrations down to a temperature of 25 mK, and we obtained a low number of about 20 phonons at the equilibrium bath temperature. The mechanical properties of Al were excellent after FIB cutting, and we recorded a quality factor of Q ∼ 3 × 10(5) for a 67 MHz resonator at a temperature of 25 mK. Between 0.2 and 2 K we find that the dissipation is linearly proportional to the temperature.