NNeurite: artificial neuronal networks for the unsupervised extraction of axonal and dendritic time-lapse signals

Fluorescence microscopy of Ca2+ transients in small neurites of the behaving mouse provides an unprecedented view of the micrometer-scale mechanisms supporting neuronal communication and computation, and therefore opens the way to understanding their role in cognition. However, the exploitation of this growing and precious experimental data is impeded by the scarcity of methods dedicated to the analysis of images of neurites activity in vivo. We present NNeurite, a set of mathematical and computational techniques specialized for the analysis of time-lapse microscopy images of neurite activity in small behaving animals. Starting from noisy and unstable microscopy images containing an unknown number of small neurites, NNeurite simultaneously aligns images, denoises signals and extracts the location and the temporal activity of the sources of Ca2+ transients. At the core of NNeurite is a novel artificial neuronal network(NN) which we have specifically designed to solve the non-negative matrix factorization (NMF)problem modeling source separation in fluorescence microscopy images. For the first time, we have embedded non-rigid image alignment in the NMF optimization procedure, hence allowing to stabilize images based on the transient and weak neurite signals. NNeurite processing is free of any human intervention as NN training is unsupervised and the unknown number of Ca2+ sources is automatically obtained by the NN-based computation of a low-dimensional representation of time-lapse images. Importantly, the spatial shapes of the sources of Ca2+ fluorescence are not constrained in NNeurite, which allowed to automatically extract the micrometer-scale details of dendritic and axonal branches, such dendritic spines and synaptic boutons, in the cortex of behaving mice. We provide NNeurite as a free and open-source library to support the efforts of the community in advancing in vivo microscopy of neurite activity.

Light activation of 3D-printed structures: from millimeter to sub-micrometer scale

Three-dimensional (3D) printing enables the fabrication of complex, highly customizable structures, which are difficult to fabricate using conventional fabrication methods. Recently, the concept of four-dimensional (4D) printing has emerged, which adds active and responsive functions to 3D-printed structures. Deployable or adaptive structures with desired structural and functional changes can be fabricated using 4D printing; thus, 4D printing can be applied to actuators, soft robots, sensors, medical devices, and active and reconfigurable photonic devices. The shape of 3D-printed structures can be transformed in response to external stimuli, such as heat, light, electric and magnetic fields, and humidity. Light has unique advantages as a stimulus for active devices because it can remotely and selectively induce structural changes. There have been studies on the light activation of nanomaterial composites, but they were limited to rather simple planar structures. Recently, the light activation of 3D-printed complex structures has attracted increasing attention. However, there has been no comprehensive review of this emerging topic yet. In this paper, we present a comprehensive review of the light activation of 3D-printed structures. First, we introduce representative smart materials and general shape-changing mechanisms in 4D printing. Then, we focus on the design and recent demonstration of remote light activation, particularly detailing photothermal activations based on nanomaterial composites. We explain the light activation of 3D-printed structures from the millimeter to sub-micrometer scale.

Chemical and elemental mapping of spent nuclear fuel sections by soft X-ray spectromicroscopy

Soft X-ray spectromicroscopy at the O K-edge, U N 4,5-edges and Ce M 4,5-edges has been performed on focused ion beam sections of spent nuclear fuel for the first time, yielding chemical information on the sub-micrometer scale. To analyze these data, a modification to non-negative matrix factorization (NMF) was developed, in which the data are no longer required to be non-negative, but the non-negativity of the spectral components and fit coefficients is largely preserved. The modified NMF method was utilized at the O K-edge to distinguish between two components, one present in the bulk of the sample similar to UO2 and one present at the interface of the sample which is a hyperstoichiometric UO2+x species. The species maps are consistent with a model of a thin layer of UO2+x over the entire sample, which is likely explained by oxidation after focused ion beam (FIB) sectioning. In addition to the uranium oxide bulk of the sample, Ce measurements were also performed to investigate the oxidation state of that fission product, which is the subject of considerable interest. Analysis of the Ce spectra shows that Ce is in a predominantly trivalent state, with a possible contribution from tetravalent Ce. Atom probe analysis was performed to provide confirmation of the presence and localization of Ce in the spent fuel.

Global cerebrospinal fluid circulation mapping using gold nanoparticle enhanced X-ray microtomography reveals region-specific brain and spinal cord CSF pathways

Cerebrospinal fluid (CSF) movement within the brain interstitium is essential for the development and functioning of the brain. However, the interstitium has largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with CSF. Here, we developed a novel technique for CSF tracking, gold nanoparticle enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF pathways during development. Using this method and subsequent histological analysis, we map global CSF pathways and present novel particle size-dependent circulation patterns through the CNS. We identify an intraparenchymal CSF circulation that targets stem cell-rich and cholinergic neuronal populations. CSF solute distribution to these areas is mediated by CSF flow along projections from the basal cisterns which is altered in posthemorrhagic hydrocephalus. Our study uncovers region-specific patterns in a biologically driven CSF circulation that has implications for normal brain development and the pathophysiology of hydrocephalus and neurodegenerative disorders.

Reconfigurable Surface Micropatterns Based on the Magnetic Field-Induced Shape Memory Effect in Magnetoactive Elastomers

A surface relief grating with a period of 30 µm is embossed onto the surface of magnetoactive elastomer (MAE) samples in the presence of a moderate magnetic field of about 180 mT. The grating, which is represented as a set of parallel stripes with two different amplitude reflectivity coefficients, is detected via diffraction of a laser beam in the reflection configuration. Due to the magnetic-field-induced plasticity effect, the grating persists on the MAE surface for at least 90 h if the magnetic field remains present. When the magnetic field is removed, the diffraction efficiency vanishes in a few minutes. The described effect is much more pronounced in MAE samples with larger content of iron filler (80 wt%) than in the samples with lower content of iron filler (70 wt%). A simple theoretical model is proposed to describe the observed dependence of the diffraction efficiency on the applied magnetic field. Possible applications of MAEs as magnetically reconfigurable diffractive optical elements are discussed. It is proposed that the described experimental method can be used as a convenient tool for investigations of the dynamics of magnetically induced plasticity of MAEs on the micrometer scale.

Microtopographical guidance of macropinocytic signaling patches

In fast-moving cells such as amoeba and immune cells, dendritic actin filaments are spatiotemporally regulated to shape large-scale plasma membrane protrusions. Despite their importance in migration, as well as in particle and liquid ingestion, how their dynamics are affected by micrometer-scale features of the contact surface is still poorly understood. Here, through quantitative image analysis of Dictyostelium on microfabricated surfaces, we show that there is a distinct mode of topographical guidance directed by the macropinocytic membrane cup. Unlike other topographical guidance known to date that depends on nanometer-scale curvature sensing protein or stress fibers, the macropinocytic membrane cup is driven by the Ras/PI3K/F-actin signaling patch and its dependency on the micrometer-scale topographical features, namely PI3K/F-actin–independent accumulation of Ras-GTP at the convex curved surface, PI3K-dependent patch propagation along the convex edge, and its actomyosin-dependent constriction at the concave edge. Mathematical model simulations demonstrate that the topographically dependent initiation, in combination with the mutually defining patch patterning and the membrane deformation, gives rise to the topographical guidance. Our results suggest that the macropinocytic cup is a self-enclosing structure that can support liquid ingestion by default; however, in the presence of structured surfaces, it is directed to faithfully trace bent and bifurcating ridges for particle ingestion and cell guidance.

Observation of a flat band and bandgap in millimeter-scale twisted bilayer graphene

Magic-angle twisted bilayer graphene, consisting of two graphene layers stacked at a special angle, exhibits superconductivity due to the maximized density of states at the energy of the flat band. Generally, experiments on twisted bilayer graphene have been performed using micrometer-scale samples. Here we report the fabrication of twisted bilayer graphene with an area exceeding 3 × 5 mm2 by transferring epitaxial graphene onto another epitaxial graphene, and observation of a flat band and large bandgap using angle-resolved photoemission spectroscopy. Our results suggest that the substrate potential induces both the asymmetrical doping in large angle twisted bilayer graphene and the electron doped nature of the flat band in magic-angle twisted bilayer graphene.

Shearing Characteristics of Cu-Cu Joints Fabricated by Two-Step Process Using Highly -Oriented Nanotwinned Cu

Cu-Cu bonding has the potential to break through the extreme boundary of scaling down chips’ I/Os into the sub-micrometer scale. In this study, we investigated the effect of 2-step bonding on the shear strength and electrical resistance of Cu-Cu microbumps using highly <111>-oriented nanotwinned Cu (nt-Cu). Alignment and bonding were achieved at 10 s in the first step, and a post-annealing process was further conducted to enhance its bonding strength. Results show that bonding strength was enhanced by 2–3 times after a post-annealing step. We found 50% of ductile fractures among 4548 post-annealed microbumps in one chip, while the rate was less than 20% for the as-bonded counterparts. During the post-annealing, interfacial grain growth and recrystallization occurred, and the bonding interface was eliminated. Ductile fracture in the form of zig-zag grain boundary was found at the original bonding interface, thus resulting in an increase in bonding strength of the microbumps.

Microvalve with Trapezoid-Shaped Cross-Section for Deep Microchannels

Simple microfluidic systems for handling large particles such as three-dimensional (3D) cultured cells, microcapsules, and animalcules have contributed to the advancement of biology. However, obtaining a highly integrated microfluidic device for handling large particles is difficult because there are no suitable microvalves for deep microchannels. Therefore, this study proposes a microvalve with a trapezoid-shaped cross-section to close a deep microchannel. The proposed microvalve can close a 350 μm deep microchannel, which is suitable for handling hundreds of micrometer-scale particles. A double-inclined lithography process was used to fabricate the trapezoid-shaped cross-section. The microvalve was fabricated by bonding three polydimethylsiloxane layers: a trapezoid-shaped liquid channel layer, a membrane, and a pneumatic channel layer. The pneumatic balloon, consisting of the membrane and the pneumatic channel, was located beneath a trapezoid-shaped cross-section microchannel. The valve was operated by the application of pneumatic pressure to the pneumatic channel. We experimentally confirmed that the expansion of the pneumatic balloon could close the 350 μm deep microchannel.