Cultured mammalian cells are a common model system for the study of epithelial biology and mechanics. Epithelia are often considered as pseudo-two dimensional and thus imaged and analyzed with respect to the apical tissue surface. We found that the three-dimensional architecture of epithelial monolayers can vary widely even within small culture wells, and that layers that appear organized in the plane of the tissue can show gross disorganization in the apical-basal plane. Epithelial cell shapes should be analyzed in 3D to understand the architecture and maturity of the cultured tissue to accurately compare between experiments. Here, we present a detailed protocol for the use of our image analysis pipeline, Automated Layer Analysis (ALAn), developed to quantitatively characterize the architecture of cultured epithelial layers. ALAn is based on a set of rules that are applied to the spatial distributions of DNA and actin signals in the apical-basal (depth) dimension of cultured layers obtained from imaging cultured cell layers using a confocal microscope. ALAn facilitates reproducibility across experiments, investigations, and labs, providing users with quantitative, unbiased characterization of epithelial architecture and maturity. Key features • This protocol was developed to spatially analyze epithelial monolayers in an automated and unbiased fashion. • ALAn requires two inputs: the spatial distributions of nuclei and actin in cultured cells obtained using confocal fluorescence microscopy. • ALAn code is written in Python3 using the Jupyter Notebook interactive format. • Optimized for use in Marbin-Darby Canine Kidney (MDCK) cells and successfully applied to characterize human MCF-7 mammary gland-derived and Caco-2 colon carcinoma cells. • This protocol utilizes Imaris software to segment nuclei but may be adapted for an alternative method. ALAn requires the centroid coordinates and volume of nuclei.

Two-dimensional transition metal dichalcogenides (TMDCs) have natural advantages in overcoming the short-channel effect in field-effect transistors (FETs) and in fabricating three-dimensional FETs, which benefit in increasing device density. However, so far, most reported works related to MoS2 FETs with a sub-100 nm channel employ mechanically exfoliated materials and all of the works involve electron beam lithography (EBL), which may limit their application in fabricating wafer-scale device arrays as demanded in integrated circuits (ICs). In this work, MoS2 FET arrays with a side-wall source and drain electrodes vertically distributed are designed and fabricated. The channel length of the as-fabricated FET is basically determined by the thickness of an insulating layer between the source and drain electrodes. The vertically distributed source and drain electrodes enable to reduce the electrode-occupied area and increase in the device density. The as-fabricated vertical FETs exhibit on/off ratios comparable to those of mechanically exfoliated MoS2 FETs with a nanoscale channel length under identical VDS. In addition, the as-fabricated FETs can work at a VDS as low as 10 mV with a desirable on/off ratio (1.9 × 107), which benefits in developing low-power devices. Moreover, the fabrication process is free from EBL and can be applied to wafer-scale device arrays. The statistical results show that the fabricated FET arrays have a device yield of 87.5% and an average on/off ratio of about 1.7 × 106 at a VDS of 10 mV, with the lowest and highest ones to be about 1.3 × 104 and 1.9 × 107, respectively, demonstrating the good reliability of our fabrication process. Our work promises a bright future for TMDCs in realizing high-density and low-power nanoelectronic devices in ICs.

The small intestine is a complex organ with a characteristic architecture and a major site for drug and nutrient absorption. The three-dimensional (3D) topography organized in finger-like protrusions called villi increases surface area remarkably, granting a more efficient absorption process. The intestinal mucosa, where this process occurs, is a multilayered and multicell-type tissue barrier. In vitro intestinal models are routinely used to study different physiological and pathological processes in the gut, including compound absorption. Still, standard models are typically two-dimensional (2D) and represent only the epithelial barrier, lacking the cues offered by the 3D architecture and the stromal components present in vivo, often leading to inaccurate results. In this work, we studied the impact of the 3D architecture of the gut on drug transport using a bioprinted 3D model of the intestinal mucosa containing both the epithelial and the stromal compartments. Human intestinal fibroblasts were embedded in a previously optimized hydrogel bioink, and enterocytes and goblet cells were seeded on top to mimic the intestinal mucosa. The embedded fibroblasts thrived inside the hydrogel, remodeling the surrounding extracellular matrix. The epithelial cells fully covered the hydrogel scaffolds and formed a uniform cell layer with barrier properties close to in vivo. In particular, the villus-like model revealed overall increased permeability compared to a flat counterpart composed by the same hydrogel and cells. In addition, the efflux activity of the P-glycoprotein (P-gp) transporter was significantly reduced in the villus-like scaffold compared to a flat model, and the genetic expression of other drugs transporters was, in general, more relevant in the villus-like model. Globally, this study corroborates that the presence of the 3D architecture promotes a more physiological differentiation of the epithelial barrier, providing more accurate data on drug absorbance measurements.

Despite more than a century of intensive study of mitotic chromosomes, their three-dimensional organization remains enigmatic. The last decade established Hi-C as a method of choice for study of spatial genome-wide interactions. Although its utilization has been focused mainly on studying genomic interactions in interphase nuclei, the method can be also successfully applied to study 3D architecture and genome folding in mitotic chromosomes. However, obtaining sufficient number of mitotic chromosomes as an input material and effective coupling with Hi-C method is challenging in plant species. An elegant way to overcome hindrances with obtaining a pure mitotic chromosome fraction is their isolation via flow cytometric sorting. This chapter presents a protocol describing plant sample preparation for chromosome conformation studies, for flow-sorting of plant mitotic metaphase chromosomes and for the Hi-C procedure.

The widespread use of chemicals has brought serious water pollution threatening human health and environment, which requires green, fast, and low-cost purification urgently. Here, we build up a novel material family of sky-parking-like 3D structured graphene oxides (SP-GOs) with adjustable interlayer-space of 0.8-1.7 nm via the insertion of different sized diamine compounds as support pillars between GO layers. The assembled 3D SP-GOs exhibit superior adsorption capacity and short removal time for various aromatic organic compounds in water, achieving record-breaking maximum adsorption capacity of 535.79 mg g-1 toward the most common water-pollutant bisphenol A (BPA) at ambient conditions as well as significantly improved removal of other organic pollutants including sulfapyridine, carbamazepine, ketoprofen and 2-naphthol. The construction of SP-GO provides a simple approach for evolving the GO material from 2D to 3D and a new avenue for the decontamination of pollutants in environmental remediation.

Exploring capable and universal electrode materials could promote the development of alkalis (Li, Na, K) ion batteries. 2D MXene material is an ideal host for the alkalis (Li, Na, K) ion storage, but its electrochemical performance is limited by serious re-stacking and aggregation problems. Herein, we cleverly combined electrostatic self-assembly with gas-phase vulcanization method to successfully combine Ti3C2Tx-MXene with ultra-long recyclability and high conductivity with MnS, which presents high specific capacity but poor conductivity. The as-prepared 3D hierarchical Ti3C2Tx/MnS composites have an unique sandwich-like constituent units. The tiny MnS nanoparticles are restricted between the Ti3C2Tx layers and play a key role in expanding the Ti3C2Tx interlayer spacing. As a result, the 3D Ti3C2Tx/MnS composites as the anode of LIBs exhibits a superior capacities of 826 and 634 mAh/g after 1000 and 3000 cycles at 0.5 and 1.0 A/g, respectively. More importantly, we reveal the reaction mechanism that the specific capacity first increases and then gradually stabilizes with the increase of charge and discharge cycle times when the as-prepared 3D Ti3C2Tx/MnS was used as the anode of LIBs. In addition, we have also used this material in SIBs and PIBs and achieved remarkable electrochemical capability, with a specific capacity of 107 mAh/g after 2500 cycles at 0.5 A/g or 127 mAh/g after the 2000th cycle at 0.2 A/g, respectively.

Land plants have two types of shoot-supporting systems, root system and rhizoid system, in vascular plants and bryophytes. However, since the evolutionary origin of the systems is different, how much they exploit common systems or distinct systems to architect their structures is largely unknown. To understand the regulatory mechanism of how bryophytes architect the rhizoid system responding to environmental factors, we have developed the methodology to visualize and quantitatively analyze the rhizoid system of the moss, Physcomitrium patens, in 3D. The rhizoids having a diameter of 21.3 µm on the average were visualized by refraction-contrast X-ray micro-computed tomography using coherent X-ray optics available at synchrotron radiation facility SPring-8. Three types of shape (ring-shape, line and black circle) observed in tomographic slices of specimens embedded in paraffin were confirmed to be the rhizoids by optical and electron microscopy. Comprehensive automatic segmentation of the rhizoids, which appeared in three different form types in tomograms, was tested by a method using a Canny edge detector or machine learning. The accuracy of output images was evaluated by comparing with the manually segmented ground truth images using measures such as F1 score and Intersection over Union, revealing that the automatic segmentation using machine learning was more effective than that using the Canny edge detector. Thus, machine learning-based skeletonized 3D model revealed quite dense distribution of rhizoids. We successfully visualized the moss rhizoid system in 3D for the first time.

The pseudocapacitive properties of CeO2 are largely dependent on its surface Faradaic redox reaction kinetics; however, its electrochemical performance is still limited by the low utilization due to the inefficient diffusionfreeways and the limited active sites. Herein, we prepare a 0D/3D composite composed of oxygen-deficient CeO2 quantum dots (0D) anchored on a 3D hollow porous N-doped carbon framework (CeO2-x QD@PHC) via a facile template-confined strategy followed by a chemical co-precipitation. The refined QDs and hollow structure greatly shorten the ion diffusion paths and lower the internal strain during cycling. The integration of CeO2-x QDs with PHC structure endows enriched accessible active sites and enhances the electrical properties. As a result, the optimized CeO2-x QD@PHC exhibits an improved specific capacitance and good rate performance in comparison to those of the CeO2-x-free PHC. Moreover, a symmetric supercapacitor with CeO2-x QD@PHC as an electrode is constructed, delivering a high energy density of 3.874 Wh kg-1 at a power density of 149.98 W kg-1.

Two-dimensional (2D) MXene materials show great potential in energy storage devices. However, the self-restacking of MXene nanosheets and the sluggish lithium-ion (Li+) kinetics greatly hinder their rate capability and cycling stability. Herein, we interlink 2D V2CTx MXene nanosheets with rGO to construct a 3D porous V2CTx-rGO composite. X-ray spectroscopy study reveals the close interfacial contact between V2CTx and rGO via electron transfer from V to C atoms. Benefiting from the close combination and optimized ion transport channel, V2CTx-rGO offers a high-rate Li+ storage performance and excellent cycling stability over 2000 cycles with negligible capacity attenuation. Moreover, it exhibits a dominant mechanism of intercalation pseudocapacitance and efficient Li+ transport proved by density functional theory calculation. This rationally designed 3D V2CTx-rGO has implications for the study of the MXene composite\'s structure and energy storage devices with high rate and stability.

Advanced materials with aligned cellulose nanocrystals (CNCs) have attracted much attention due to their remarkable mechanical and optical properties, but most of them still focus on 1D or 2D architectures. Herein, complex 3D architectures as pseudo catenoid hollow xerogels with aligned CNCs are prepared from dynamic hydrogels by mechanical stretching and air-drying process. Aligned CNCs endow the pseudo catenoids with distinct birefringence in addition to reinforcement. The mechanical properties of pseudo catenoid architecture are revealed for the first time to be controlled at two stages on diverse length scales. Both the aligned CNCs on the nanoscale and the geometry of the xerogels affect the mechanical properties. The inwardly curved surface of the pseudo catenoid xerogel makes the structure conducive to energy dissipation. These both stages of controls on the mechanical properties can be adjusted by changing the morphology of the initial hydrogels and the mechanical stretching ratios. These results will provide a new perspective for the design and manufacture advanced materials with tailored mechanical properties and functions.