Label-Free Techniques for Probing Biomolecular Condensates.

Biomolecular condensates play important roles in a wide array of fundamental biological processes, such as cellular compartmentalization, cellular regulation, and other biochemical reactions. Since their discovery and first observations, an extensive and expansive library of tools has been developed to investigate various aspects and properties, encompassing structural and compositional information, material properties, and their evolution throughout the life cycle from formation to eventual dissolution. This Review presents an overview of the expanded set of tools and methods that researchers use to probe the properties of biomolecular condensates across diverse scales of length, concentration, stiffness, and time. In particular, we review recent years' exciting development of label-free techniques and methodologies. We broadly organize the set of tools into 3 categories: (1) imaging-based techniques, such as transmitted-light microscopy (TLM) and Brillouin microscopy (BM), (2) force spectroscopy techniques, such as atomic force microscopy (AFM) and the optical tweezer (OT), and (3) microfluidic platforms and emerging technologies. We point out the tools' key opportunities, challenges, and future perspectives and analyze their correlative potential as well as compatibility with other techniques. Additionally, we review emerging techniques, namely, differential dynamic microscopy (DDM) and interferometric scattering microscopy (iSCAT), that have huge potential for future applications in studying biomolecular condensates. Finally, we highlight how some of these techniques can be translated for diagnostics and therapy purposes. We hope this Review serves as a useful guide for new researchers in this field and aids in advancing the development of new biophysical tools to study biomolecular condensates.

Nanofluidic logic with mechano-ionic memristive switches.

Neuromorphic systems are typically based on nanoscale electronic devices, but nature relies on ions for energy-efficient information processing. Nanofluidic memristive devices could thus potentially be used to construct electrolytic computers that mimic the brain down to its basic principles of operation. Here we report a nanofluidic device that is designed for circuit-scale in-memory processing. The device, which is fabricated using a scalable process, combines single-digit nanometric confinement and large entrance asymmetry and operates on the second timescale with a conductance ratio in the range of 9 to 60. In operando optical microscopy shows that the memory capabilities are due to the reversible formation of liquid blisters that modulate the conductance of the device. We use these mechano-ionic memristive switches to assemble logic circuits composed of two interactive devices and an ohmic resistor.

Label-Free Imaging of DNA Interactions with 2D Materials.

Two-dimensional (2D) materials offer potential as substrates for biosensing devices, as their properties can be engineered to tune interactions between the surface and biomolecules. Yet, not many methods can measure these interactions in a liquid environment without introducing labeling agents such as fluorophores. In this work, we harness interferometric scattering (iSCAT) microscopy, a label-free imaging technique, to investigate the interactions of single molecules of long dsDNA with 2D materials. The millisecond temporal resolution of iSCAT allows us to capture the transient interactions and to observe the dynamics of unlabeled DNA binding to a hexagonal boron nitride (hBN) surface in solution for extended periods (including a fraction of 10%, of trajectories lasting longer than 110 ms). Using a focused ion beam technique to engineer defects, we find that DNA binding affinity is enhanced at defects; when exposed to long lanes, DNA binds preferentially at the lane edges. Overall, we demonstrate that iSCAT imaging is a useful tool to study how biomolecules interact with 2D materials, a key component in engineering future biosensors.

Open-source microscope add-on for structured illumination microscopy.

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

Synthesis of Fluorescent Cyclic Peptides via Gold(I)-Catalyzed Macrocyclization.

Rapid and efficient cyclization methods that form structurally novel peptidic macrocycles are of high importance for medicinal chemistry. Herein, we report the first gold(I)-catalyzed macrocyclization of peptide-EBXs (ethynylbenziodoxolones) via C2-Trp C-H activation. This reaction was carried out in the presence of protecting group free peptide sequences and is enabled by a simple commercial gold catalyst (AuCl·Me2S). The method displayed a rapid reaction rate (within 10 min), wide functional group tolerance (27 unprotected peptides were cyclized), and up to 86% isolated yield. The obtained highly conjugated cyclic peptide linker, formed through C-H alkynylation, can be directly applied to live-cell imaging as a fluorescent probe without further attachment of fluorophores.

Label-free identification of protein aggregates using deep learning.

Protein misfolding and aggregation play central roles in the pathogenesis of various neurodegenerative diseases (NDDs), including Huntington's disease, which is caused by a genetic mutation in exon 1 of the Huntingtin protein (Httex1). The fluorescent labels commonly used to visualize and monitor the dynamics of protein expression have been shown to alter the biophysical properties of proteins and the final ultrastructure, composition, and toxic properties of the formed aggregates. To overcome this limitation, we present a method for label-free identification of NDD-associated aggregates (LINA). Our approach utilizes deep learning to detect unlabeled and unaltered Httex1 aggregates in living cells from transmitted-light images, without the need for fluorescent labeling. Our models are robust across imaging conditions and on aggregates formed by different constructs of Httex1. LINA enables the dynamic identification of label-free aggregates and measurement of their dry mass and area changes during their growth process, offering high speed, specificity, and simplicity to analyze protein aggregation dynamics and obtain high-fidelity information.

Liquid-activated quantum emission from pristine hexagonal boron nitride for nanofluidic sensing.

Liquids confined down to the atomic scale can show radically new properties. However, only indirect and ensemble measurements operate in such extreme confinement, calling for novel optical approaches that enable direct imaging at the molecular level. Here we harness fluorescence originating from single-photon emitters at the surface of hexagonal boron nitride for molecular imaging and sensing in nanometrically confined liquids. The emission originates from the chemisorption of organic solvent molecules onto native surface defects, revealing single-molecule dynamics at the interface through the spatially correlated activation of neighbouring defects. Emitter spectra further offer a direct readout of the local dielectric properties, unveiling increasing dielectric order under nanometre-scale confinement. Liquid-activated native hexagonal boron nitride defects bridge the gap between solid-state nanophotonics and nanofluidics, opening new avenues for nanoscale sensing and optofluidics.

Confinement-Controlled Water Engenders Unusually High Electrochemical Capacitance.

The electrodynamics of nanoconfined water have been shown to change dramatically compared to bulk water, opening room for safe electrochemical systems. We demonstrate a nanofluidic "water-only" battery that exploits anomalously high electrolytic properties of pure water at firm confinement. The device consists of a membrane electrode assembly of carbon-based nanomaterials, forming continuously interconnected water-filled nanochannels between the separator and electrodes. The efficiency of the cell in the 1-100 nm pore size range shows a maximum energy density at 3 nm, challenging the region of the current metal-ion batteries. Our results establish the electrodynamic fundamentals of nanoconfined water and pave the way for low-cost and inherently safe energy storage solutions that are much needed in the renewable energy sector.

Selective Growth of van der Waals Heterostructures Enabled by Electron-Beam Irradiation.

Van der Waals heterostructures (vdWHSs) enable the fabrication of complex electronic devices based on two-dimensional (2D) materials. Ideally, these vdWHSs should be fabricated in a scalable and repeatable way and only in the specific areas of the substrate to lower the number of technological operations inducing defects and impurities. Here, we present a method of selective fabrication of vdWHSs via chemical vapor deposition by electron-beam (EB) irradiation. We distinguish two growth modes: positive (2D materials nucleate on the irradiated regions) on graphene and tungsten disulfide (WS2) substrates, and negative (2D materials do not nucleate on the irradiated regions) on the graphene substrate. The growth mode is controlled by limiting the air exposure of the irradiated substrate and the time between irradiation and growth. We conducted Raman mapping, Kelvin-probe force microscopy, X-ray photoelectron spectroscopy, and density-functional theory modeling studies to investigate the selective growth mechanism. We conclude that the selective growth is explained by the competition of three effects: EB-induced defects, adsorption of carbon species, and electrostatic interaction. The method here is a critical step toward the industry-scale fabrication of 2D-materials-based devices.

Nature-Inspired Stalactite Nanopores for Biosensing and Energy Harvesting.

Nature provides a wide range of self-assembled structures from the nanoscale to the macroscale. Under the right thermodynamic conditions and with the appropriate material supply, structures like stalactites, icicles, and corals can grow. However, the natural growth process is time-consuming. This work demonstrates a fast, nature-inspired method for growing stalactite nanopores using heterogeneous atomic deposition of hafnium dioxide at the orifice of templated silicon nitride apertures. The stalactite nanostructures combine the benefits of reduced sensing region typically for 2-dimensional material nanopores with the asymmetric geometry of capillaries, resulting in ionic selectivity, stability, and scalability. The proposed growing method provides an adaptable nanopore platform for basic and applied nanofluidic research, including biosensing, energy science, and filtration technologies.

Nanoscale thermal control of a single living cell enabled by diamond heater-thermometer.

We report a new approach to controllable thermal stimulation of a single living cell and its compartments. The technique is based on the use of a single polycrystalline diamond particle containing silicon-vacancy (SiV) color centers. Due to the presence of amorphous carbon at its intercrystalline boundaries, such a particle is an efficient light absorber and becomes a local heat source when illuminated by a laser. Furthermore, the temperature of such a local heater is tracked by the spectral shift of the zero-phonon line of SiV centers. Thus, the diamond particle acts simultaneously as a heater and a thermometer. In the current work, we demonstrate the ability of such a Diamond Heater-Thermometer (DHT) to locally alter the temperature, one of the numerous parameters that play a decisive role for the living organisms at the nanoscale. In particular, we show that the local heating of 11-12 °C relative to the ambient temperature (22 °C) next to individual HeLa cells and neurons, isolated from the mouse hippocampus, leads to a change in the intracellular distribution of the concentration of free calcium ions. For individual HeLa cells, a long-term (about 30 s) increase in the integral intensity of Fluo-4 NW fluorescence by about three times is observed, which characterizes an increase in the [Ca2+]cyt concentration of free calcium in the cytoplasm. Heating near mouse hippocampal neurons also caused a calcium surge-an increase in the intensity of Fluo-4 NW fluorescence by 30% and a duration of ~ 0.4 ms.

The Three-Phase Contact Potential Difference Modulates the Water Surface Charge.

The surface charge of an open water surface is crucial for solvation phenomena and interfacial processes in aqueous systems. However, the magnitude of the charge is controversial, and the physical mechanism of charging remains incompletely understood. Here we identify a previously overlooked physical mechanism determining the surface charge of water. Using accurate charge measurements of water microdrops, we demonstrate that the water surface charge originates from the electrostatic effects in the contact line vicinity of three phases, one of which is water. Our experiments, theory, and simulations provide evidence that a junction of two aqueous interfaces (e.g., liquid-solid and liquid-air) develops a pH-dependent contact potential difference Δϕ due to the longitudinal charge redistribution between two contacting interfaces. This universal static charging mechanism may have implications for the origin of electrical potentials in biological, nanofluidic, and electrochemical systems and helps to predict and control the surface charge of water in various experimental environments.

Fluorescence Microscopy: a statistics-optics perspective.

Fundamental properties of light unavoidably impose features on images collected using fluorescence microscopes. Modeling these features is ever more important in quantitatively interpreting microscopy images collected at scales on par or smaller than light's wavelength. Here we review the optics responsible for generating fluorescent images, fluorophore properties, microscopy modalities leveraging properties of both light and fluorophores, in addition to the necessarily probabilistic modeling tools imposed by the stochastic nature of light and measurement.

Optical imaging of the small intestine immune compartment across scales.

The limitations of 2D microscopy constrain our ability to observe and understand tissue-wide networks that are, by nature, 3-dimensional. Optical projection tomography (OPT) enables the acquisition of large volumes (ranging from micrometres to centimetres) in various tissues. We present a multi-modal workflow for the characterization of both structural and quantitative parameters of the mouse small intestine. As proof of principle, we evidence its applicability for imaging the mouse intestinal immune compartment and surrounding mucosal structures. We quantify the volumetric size and spatial distribution of Isolated Lymphoid Follicles (ILFs) and quantify the density of villi throughout centimetre-long segments of intestine. Furthermore, we exhibit the age and microbiota dependence for ILF development, and leverage a technique that we call reverse-OPT for identifying and homing in on regions of interest. Several quantification capabilities are displayed, including villous density in the autofluorescent channel and the size and spatial distribution of the signal of interest at millimetre-scale volumes. The concatenation of 3D imaging with reverse-OPT and high-resolution 2D imaging allows accurate localisation of ROIs and adds value to interpretations made in 3D. Importantly, OPT may be used to identify sparsely-distributed regions of interest in large volumes whilst retaining compatibility with high-resolution microscopy modalities, including confocal microscopy. We believe this pipeline to be approachable for a wide-range of specialties, and to provide a new method for characterisation of the mouse intestinal immune compartment.

Substitutional p-Type Doping in NbS2 -MoS2 Lateral Heterostructures Grown by MOCVD.

Monolayer MoS2 has attracted significant attention owing to its excellent performance as an n-type semiconductor from the transition metal dichalcogenide (TMDC) family. It is however strongly desired to develop controllable synthesis methods for 2D p-type MoS2 , which is crucial for complementary logic applications but remains difficult. In this work, high-quality NbS2 -MoS2 lateral heterostructures are synthesized by one-step metal-organic chemical vapor deposition (MOCVD) together with monolayer MoS2 substitutionally doped by Nb, resulting in a p-type doped behavior. The heterojunction shows a p-type transfer characteristic with a high on/off current ratio of ≈104 , exceeding previously reported values. The band structure through the NbS2 -MoS2 heterojunction is investigated by density functional theory (DFT) and quantum transport simulations. This work provides a scalable approach to synthesize substitutionally doped TMDC materials and provides an insight into the interface between 2D metals and semiconductors in lateral heterostructures, which is imperative for the development of next-generation nanoelectronics and highly integrated devices.

High durability and stability of 2D nanofluidic devices for long-term single-molecule sensing.

Nanopores in two-dimensional (2D) membranes hold immense potential in single-molecule sensing, osmotic power generation, and information storage. Recent advances in 2D nanopores, especially on single-layer MoS2, focus on the scalable growth and manufacturing of nanopore devices. However, there still remains a bottleneck in controlling the nanopore stability in atomically thin membranes. Here, we evaluate the major factors responsible for the instability of the monolayer MoS2 nanopores. We identify chemical oxidation and delamination of monolayers from their underlying substrates as the major reasons for the instability of MoS2 nanopores. Surface modification of the substrate and reducing the oxygen from the measurement solution improves nanopore stability and dramatically increases their shelf-life. Understanding nanopore growth and stability can provide insights into controlling the pore size, shape and can enable long-term measurements with a high signal-to-noise ratio and engineering durable nanopore devices.

Wafer-scale MoS2 with water-vapor assisted showerhead MOCVD.

Among numerous thin film synthesis methods, metalorganic chemical vapor deposition performed in a showerhead reactor is the most promising one for broad use in scalable and commercially adaptable two-dimensional material synthesis processes. Adapting the most efficient monolayer growth methodologies from tube-furnace systems to vertical-showerhead geometries allows us to overcome the intrinsic process limitations and improve the overall monolayer yield quality. Here, we demonstrate large-area, monolayer molybdenum disulphide growth by combining gas-phase precursor supply with unique tube-furnace approaches of utilizing sodium molybdate pre-seeding solution spincoated on a substrate along with water vapor added during the growth step. The engineered process yields a high-quality, 4-inch scale monolayer film on sapphire wafers. The monolayer growth coverage, average crystal size and defect density were evaluated using Raman and photoluminescence spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and scanning transmission electron microscopy imaging. Our findings provide a direct step forward toward developing a reproducible and large-scale MoS2 synthesis with commercial showerhead reactors.

High-Throughput Nanopore Fabrication and Classification Using Xe-Ion Irradiation and Automated Pore-Edge Analysis.

Large-area nanopore drilling is a major bottleneck in state-of-the-art nanoporous 2D membrane fabrication protocols. In addition, high-quality structural and statistical descriptions of as-fabricated porous membranes are key to predicting the corresponding membrane-wide permeation properties. In this work, we investigate Xe-ion focused ion beam as a tool for scalable, large-area nanopore fabrication on atomically thin, free-standing molybdenum disulfide. The presented irradiation protocol enables designing ultrathin membranes with tunable porosity and pore dimensions, along with spatial uniformity across large-area substrates. Fabricated nanoporous membranes are then characterized using scanning transmission electron microscopy imaging, and the observed nanopore geometries are analyzed through a pore-edge detection and analysis script. We further demonstrate that the obtained structural and statistical data can be readily passed on to computational and analytical tools to predict the permeation properties at both individual pore and membrane-wide scales. As an example, membranes featuring angstrom-scale pores are investigated in terms of their emerging water and ion flow properties through extensive all-atom molecular dynamics simulations. We believe that the combination of experimental and analytical approaches presented here will yield accurate physics-based property estimates and thus potentially enable a true function-by-design approach to fabrication for applications such as osmotic power generation and desalination/filtration.

Stress induced delamination of suspended MoS2 in aqueous environments.

Applying hydrostatic pressure with suspended 2D material thin membranes allows probing the effects of lateral strain on the ion and fluid transport through nanopores. We demonstrate how both permanent and temporary delamination of 2D materials can be induced by pressure and potential differences between the membrane, causing a strong mechanosensitive modulation of ion transport. Our methodology is based on in situ measurements of ionic current and streaming modulation as the supporting membrane is indented or bulged with pressure. We demonstrate how indirect measurements of fluid transport through delaminated MoS2 membranes is achieved through monitoring streaming current and potential. This is combined with TEM images of 2D material membranes before and after aqueous measurements, showing temporary delamination during mechanical or electrical stress. The obtained results allow a better understanding of measurements with supported 2D materials, i.e. avoiding misinterpreting measured data, and could be used to probe how the electrical field and fluid flow at the nanoscale influence the adhesion of supported 2D materials.

Engineering Optically Active Defects in Hexagonal Boron Nitride Using Focused Ion Beam and Water.

Hexagonal boron nitride (hBN) has emerged as a promising material platform for nanophotonics and quantum sensing, hosting optically active defects with exceptional properties such as high brightness and large spectral tuning. However, precise control over deterministic spatial positioning of emitters in hBN remained elusive for a long time, limiting their proper correlative characterization and applications in hybrid devices. Recently, focused ion beam (FIB) systems proved to be useful to engineer several types of spatially defined emitters with various structural and photophysical properties. Here we systematically explore the physical processes leading to the creation of optically active defects in hBN using FIB and find that beam-substrate interaction plays a key role in the formation of defects. These findings are confirmed using transmission electron microscopy, which reveals local mechanical deterioration of the hBN layers and local amorphization of ion beam irradiated hBN. Additionally, we show that, upon exposure to water, amorphized hBN undergoes a structural and optical transition between two defect types with distinctive emission properties. Moreover, using super-resolution optical microscopy combined with atomic force microscopy, we pinpoint the exact location of emitters within the defect sites, confirming the role of defected edges as primary sources of fluorescent emission. This lays the foundation for FIB-assisted engineering of optically active defects in hBN with high spatial and spectral control for applications ranging from integrated photonics, to nanoscale sensing, and to nanofluidics.