Dense, Continuous Membrane Labeling and Expansion Microscopy Visualization of Ultrastructure in Tissues.

Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines a novel membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validated the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supported the imaging of membranes and proteins at a resolution of ~60 nm on a confocal microscope. We demonstrated the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yielded ~35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation.

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding.

Expansion Microscopy for Beginners: Visualizing Microtubules in Expanded Cultured HeLa Cells.

Expansion microscopy (ExM) is a technique that physically expands preserved cells and tissues before microscope imaging, so that conventional diffraction-limited microscopes can perform nanoscale-resolution imaging. In ExM, biomolecules or their markers are linked to a dense, swellable gel network synthesized throughout a specimen. Mechanical homogenization of the sample (e.g., by protease digestion) and the addition of water enable isotropic swelling of the gel, so that the relative positions of biomolecules are preserved. We previously presented ExM protocols for analyzing proteins and RNAs in cells and tissues. Here we describe a cookbook-style ExM protocol for expanding cultured HeLa cells with immunostained microtubules, aimed to help newcomers familiarize themselves with the experimental setups and skills required to successfully perform ExM. Our aim is to help beginners, or students in a wet-lab classroom setting, learn all the key steps of ExM. © 2020 The Authors.

Iterative expansion microscopy.

We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ∼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens ∼4.5 × 4.5, or ∼20×, and enables ∼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.

Monolithic 3D CMOS Using Layered Semiconductors.

Monolithic 3D integrated circuits using transition metal dichalcogenide materials and low-temperature processing are reported. A variety of digital and analog circuits are implemented on two sequentially integrated layers of devices. Inverter circuit operation at an ultralow supply voltage of 150 mV is achieved, paving the way to high-density, ultralow-voltage, and ultralow-power applications.

MoS2 Heterojunctions by Thickness Modulation.

In this work, we report lateral heterojunction formation in as-exfoliated MoS2 flakes by thickness modulation. Kelvin probe force microscopy is used to map the surface potential at the monolayer-multilayer heterojunction, and consequently the conduction band offset is extracted. Scanning photocurrent microscopy is performed to investigate the spatial photocurrent response along the length of the device including the source and the drain contacts as well as the monolayer-multilayer junction. The peak photocurrent is measured at the monolayer-multilayer interface, which is attributed to the formation of a type-I heterojunction. The work presents experimental and theoretical understanding of the band alignment and photoresponse of thickness modulated MoS2 junctions with important implications for exploring novel optoelectronic devices.

Engineering light outcoupling in 2D materials.

When light is incident on 2D transition metal dichalcogenides (TMDCs), it engages in multiple reflections within underlying substrates, producing interferences that lead to enhancement or attenuation of the incoming and outgoing strength of light. Here, we report a simple method to engineer the light outcoupling in semiconducting TMDCs by modulating their dielectric surroundings. We show that by modulating the thicknesses of underlying substrates and capping layers, the interference caused by substrate can significantly enhance the light absorption and emission of WSe2, resulting in a ∼11 times increase in Raman signal and a ∼30 times increase in the photoluminescence (PL) intensity of WSe2. On the basis of the interference model, we also propose a strategy to control the photonic and optoelectronic properties of thin-layer WSe2. This work demonstrates the utilization of outcoupling engineering in 2D materials and offers a new route toward the realization of novel optoelectronic devices, such as 2D LEDs and solar cells.

Air stable p-doping of WSe2 by covalent functionalization.

Covalent functionalization of transition metal dichalcogenides (TMDCs) is investigated for air-stable chemical doping. Specifically, p-doping of WSe(2) via NOx chemisorption at 150 °C is explored, with the hole concentration tuned by reaction time. Synchrotron based soft X-ray absorption spectroscopy (XAS) and X-ray photoelectron spectroscopy (XPS) depict the formation of various WSe(2-x-y)O(x)N(y) species both on the surface and interface between layers upon chemisorption reaction. Ab initio simulations corroborate our spectroscopy results in identifying the energetically favorable complexes, and predicting WSe(2):NO at the Se vacancy sites as the predominant dopant species. A maximum hole concentration of ∼ 10(19) cm(-3) is obtained from XPS and electrical measurements, which is found to be independent of WSe(2) thickness. This degenerate doping level facilitates 5 orders of magnitude reduction in contact resistance between Pd, a common p-type contact metal, and WSe(2). More generally, the work presents a platform for manipulating the electrical properties and band structure of TMDCs using covalent functionalization.

Strain-induced indirect to direct bandgap transition in multilayer WSe2.

Transition metal dichalcogenides, such as MoS2 and WSe2, have recently gained tremendous interest for electronic and optoelectronic applications. MoS2 and WSe2 monolayers are direct bandgap and show bright photoluminescence (PL), whereas multilayers exhibit much weaker PL due to their indirect optical bandgap. This presents an obstacle for a number of device applications involving light harvesting or detection where thicker films with direct optical bandgap are desired. Here, we experimentally demonstrate a drastic enhancement in PL intensity for multilayer WSe2 (2-4 layers) under uniaxial tensile strain of up to 2%. Specifically, the PL intensity of bilayer WSe2 is amplified by ∼ 35× , making it comparable to that of an unstrained WSe2 monolayer. This drastic PL enhancement is attributed to an indirect to direct bandgap transition for strained bilayer WSe2, as confirmed by density functional theory (DFT) calculations. Notably, in contrast to MoS2 multilayers, the energy difference between the direct and indirect bandgaps of WSe2 multilayers is small, thus allowing for bandgap crossover at experimentally feasible strain values. Our results present an important advance toward controlling the band structure and optoelectronic properties of few-layer WSe2 via strain engineering, with important implications for practical device applications.

Air-stable surface charge transfer doping of MoS2 by benzyl viologen.

Air-stable doping of transition metal dichalcogenides is of fundamental importance to enable a wide range of optoelectronic and electronic devices while exploring their basic material properties. Here we demonstrate the use of benzyl viologen (BV), which has one of the highest reduction potentials of all electron-donor organic compounds, as a surface charge transfer donor for MoS2 flakes. The n-doped samples exhibit excellent stability in both ambient air and vacuum. Notably, we obtained a high electron sheet density of ~1.2 × 10(13) cm(-2), which corresponds to the degenerate doping limit for MoS2. The BV dopant molecules can be reversibly removed by immersion in toluene, providing the ability to control the carrier sheet density as well as selective removal of surface dopants on demand. By BV doping of MoS2 at the metal junctions, the contact resistances are shown to be reduced by a factor of >3. As a proof of concept, top-gated field-effect transistors were fabricated with BV-doped n(+) source/drain contacts self-aligned with respect to the top gate. The device architecture, resembling that of the conventional Si transistors, exhibited excellent switching characteristics with a subthreshold swing of ~77 mV/decade.

Field-effect transistors built from all two-dimensional material components.

We demonstrate field-effect transistors using heterogeneously stacked two-dimensional materials for all of the components, including the semiconductor, insulator, and metal layers. Specifically, MoS2 is used as the active channel material, hexagonal-BN as the top-gate dielectric, and graphene as the source/drain and the top-gate contacts. This transistor exhibits n-type behavior with an ON/OFF current ratio of >10(6), and an electron mobility of ∼33 cm(2)/V·s. Uniquely, the mobility does not degrade at high gate voltages, presenting an important advantage over conventional Si transistors where enhanced surface roughness scattering severely reduces carrier mobilities at high gate-fields. A WSe2-MoS2 diode with graphene contacts is also demonstrated. The diode exhibits excellent rectification behavior and a low reverse bias current, suggesting high quality interfaces between the stacked layers. In this work, all interfaces are based on van der Waals bonding, presenting a unique device architecture where crystalline, layered materials with atomically uniform thicknesses are stacked on demand, without the lattice parameter constraints. The results demonstrate the promise of using an all-layered material system for future electronic applications.

Strong interlayer coupling in van der Waals heterostructures built from single-layer chalcogenides.

Semiconductor heterostructures are the fundamental platform for many important device applications such as lasers, light-emitting diodes, solar cells, and high-electron-mobility transistors. Analogous to traditional heterostructures, layered transition metal dichalcogenide heterostructures can be designed and built by assembling individual single layers into functional multilayer structures, but in principle with atomically sharp interfaces, no interdiffusion of atoms, digitally controlled layered components, and no lattice parameter constraints. Nonetheless, the optoelectronic behavior of this new type of van der Waals (vdW) semiconductor heterostructure is unknown at the single-layer limit. Specifically, it is experimentally unknown whether the optical transitions will be spatially direct or indirect in such hetero-bilayers. Here, we investigate artificial semiconductor heterostructures built from single-layer WSe2 and MoS2. We observe a large Stokes-like shift of ∼ 100 meV between the photoluminescence peak and the lowest absorption peak that is consistent with a type II band alignment having spatially direct absorption but spatially indirect emission. Notably, the photoluminescence intensity of this spatially indirect transition is strong, suggesting strong interlayer coupling of charge carriers. This coupling at the hetero-interface can be readily tuned by inserting dielectric layers into the vdW gap, consisting of hexagonal BN. Consequently, the generic nature of this interlayer coupling provides a new degree of freedom in band engineering and is expected to yield a new family of semiconductor heterostructures having tunable optoelectronic properties with customized composite layers.

MoS2 P-type transistors and diodes enabled by high work function MoOx contacts.

The development of low-resistance source/drain contacts to transition-metal dichalcogenides (TMDCs) is crucial for the realization of high-performance logic components. In particular, efficient hole contacts are required for the fabrication of p-type transistors with MoS2, a model TMDC. Previous studies have shown that the Fermi level of elemental metals is pinned close to the conduction band of MoS2, thus resulting in large Schottky barrier heights for holes with limited hole injection from the contacts. Here, we show that substoichiometric molybdenum trioxide (MoOx, x < 3), a high work function material, acts as an efficient hole injection layer to MoS2 and WSe2. In particular, we demonstrate MoS2 p-type field-effect transistors and diodes by using MoOx contacts. We also show drastic on-current improvement for p-type WSe2 FETs with MoOx contacts over devices made with Pd contacts, which is the prototypical metal used for hole injection. The work presents an important advance in contact engineering of TMDCs and will enable future exploration of their performance limits and intrinsic transport properties.

Defects activated photoluminescence in two-dimensional semiconductors: interplay between bound, charged, and free excitons.

Point defects in semiconductors can trap free charge carriers and localize excitons. The interaction between these defects and charge carriers becomes stronger at reduced dimensionalities, and is expected to greatly influence physical properties of the hosting material. We investigated effects of anion vacancies in monolayer transition metal dichalcogenides as two-dimensional (2D) semiconductors where the vacancies density is controlled by α-particle irradiation or thermal-annealing. We found a new, sub-bandgap emission peak as well as increase in overall photoluminescence intensity as a result of the vacancy generation. Interestingly, these effects are absent when measured in vacuum. We conclude that in opposite to conventional wisdom, optical quality at room temperature cannot be used as criteria to assess crystal quality of the 2D semiconductors. Our results not only shed light on defect and exciton physics of 2D semiconductors, but also offer a new route toward tailoring optical properties of 2D semiconductors by defect engineering.

Broad-range modulation of light emission in two-dimensional semiconductors by molecular physisorption gating.

In the monolayer limit, transition metal dichalcogenides become direct-bandgap, light-emitting semiconductors. The quantum yield of light emission is low and extremely sensitive to the substrate used, while the underlying physics remains elusive. In this work, we report over 100 times modulation of light emission efficiency of these two-dimensional semiconductors by physical adsorption of O2 and/or H2O molecules, while inert gases do not cause such effect. The O2 and/or H2O pressure acts quantitatively as an instantaneously reversible "molecular gating" force, providing orders of magnitude broader control of carrier density and light emission than conventional electric field gating. Physi-sorbed O2 and/or H2O molecules electronically deplete n-type materials such as MoS2 and MoSe2, which weakens electrostatic screening that would otherwise destabilize excitons, leading to the drastic enhancement in photoluminescence. In p-type materials such as WSe2, the molecular physisorption results in the opposite effect. Unique and universal in two-dimensional semiconductors, the effect offers a new mechanism for modulating electronic interactions and implementing optical devices.