SPIFFI enables single-shot super-resolution and multidimensional imaging

W. Guo; L. Feletti; A. Rađenović 

Fluorescence super-resolution microscopy has advanced optical imaging into the nanoscale regime, transforming biological and interdisciplinary research. However, wide-field super-resolution techniques often compromise temporal resolution, thereby limiting the capture of rapid and transient biological events in living systems. Here we introduce spatial polarization-induced fluorescence fluctuation imaging (SPIFFI), a multichannel polarimetric method for single-shot super-resolution imaging and six-dimensional information extraction. By leveraging the inherently smaller point spread function under polarized detection and capturing polarization-dependent spatial fluctuations across multiplexed channels, SPIFFI achieves instant resolution enhancement from a single exposure. This capability substantially enhances the feasibility of volumetric live-cell super-resolution imaging. Moreover, SPIFFI images can integrate seamlessly with existing fluctuation-based methods for further postprocessing and resolution improvement. We demonstrate the versatility of SPIFFI through experiments on both fixed and live cells, capturing rapid subcellular dynamics and enabling high-throughput, multidimensional imaging beyond the diffraction limit. SPIFFI thus offers a practical and robust platform for real-time super-resolution imaging in biological research.

Emission dipole orientation reveals dynamic single-molecule interactions with 2D crystals at solvent interfaces

W. Guo; T. Chen; N. Ronceray; E. S. Mayner; K. Watanabe et al. 

Direct observation of individual fluorescent emitters is essential for studying quantum materials, chemical reactions, and biological systems. However, current single-molecule tracking methods only focuses on the localizations of molecules, overlooking molecular configuration and orientation. In this work, we introduce a high-throughput polarized single-molecule localization microscopy that simultaneously resolves the locations and emission dipole orientations of single fluorescent emitters with nanometer precision. Using the interface between pristine hexagonal boron nitride (h-BN) and an organic solvent as a challenging platform, we capture over 10⁵ fluorescent events and reveal distinct molecular interaction dynamics at room temperature. The measured dipole orientations align with the three-fold (C₃) rotational symmetry of the h-BN lattice, and molecular dynamics in the liquid environment can be modulated electrochemically, suggesting a route for on-demand control of quantum emitters. We also find that lateral diffusion at the solid–liquid interface is far more dynamic than that of solid-state emitters. This simultaneous tracking of molecular conformation and photophysics advances the understanding of single-molecule interactions and enables real-time sensing through two-dimensional materials. Hexagonal boron nitride substrates affect the dynamics of single molecules at solid-liquid interfaces. Here, the authors use polarized super-resolution microscopy to map the 3D orientation of emitters at interfaces, thereby demonstrating how lattice structure and electricity regulate molecular behavior.

All-water supercapacitor enabled by 1-nm clay channels

В. Г. Артемов; S. Babiy; Y. Teng; J. Ma; A. Ryzhov et al. 

Water confined to channels one nanometer thick exhibits electrochemical behavior distinct from bulk water, including enhanced protonic conductivity and large dielectric anisotropy. Here, we exploit these characteristics to design a scalable electrochemical energy storage system-a “blue capacitor”-constructed entirely from naturally abundant materials. By assembling layered clays and conductive graphene, we produce 1-nm-thick channels in which confined water acts as the sole electrolyte. We systematically study different clay types, the electrode composition, and separator thickness using complementary physicochemical and electrochemical techniques. The device operates stably up to 1.6 ± 0.1 V, achieves specific capacitances of 40 F g −1 , 97 ± 2% coulombic efficiency, and stable performance over more than 60,000 charge-discharge cycles at a voltage window of 1 V and a scan rate of 10 mA. Structural and dynamic analyses validate the device architecture, water purity, and proton transport in the nanopores. These results demonstrate that nanoconfined water can function as an electrolyte in a macroscopic electrochemical device, providing a platform for exploring sustainable aqueous energy storage systems.

Non-volatile memories based on patterned metal–semiconductor heterostructures of niobium disulfide and molybdenum disulfide

Z. Wang; G. Migliato Marega; E. Collette; M. Tripathi; H. G. Ji et al. 

The performance of transistors based on two-dimensional transition metal dichalcogenide semiconductors is restricted by the poor interface quality between two-dimensional materials and conventional three-dimensional contacts. Transition-metal-dichalcogenide-based metal–semiconductor heterostructures have been developed to enhance device performance, but finding fabrication techniques that combine high-quality growth with scalability and broad applicability remains a challenge. Here we show that a method that combines metal–organic chemical vapour deposition and sulfurization can be used to create patterned heterostructures of niobium disulfide and molybdenum disulfide at the wafer scale. The niobium disulfide–molybdenum disulfide heterostructures can be used as the active channel material of field-effect transistors and non-volatile memory devices. Compared with pristine molybdenum disulfide, the heterostructures exhibit up to nine times higher on current due to a reduced contact resistance, a maximum effective mobility of 77 cm2 V−1 s−1 and a 95.8% yield (of 144 field-effect transistors). Furthermore, our floating-gate field-effect transistors show a large programming window, precise and continuous conductance modulation, endurance over 60,000 programming pulses and an estimated retention time of around 19 years. Device simulation shows that the large programming window of the long-channel devices (around 14 V) can be maintained at scaled gate lengths below 100 nm with proper control oxide scaling.

Isotopic Fingerprints of Proton-Mediated Dielectric Relaxation in Solid and Liquid Water

A. Ryzhov; A. Andreev; A. Radenovic; V. Artemov 

We report cross-validated measurements of the isotope effect on dielectric relaxation for four isotopologues of ice and water, including the 1–105 Hz region, in which only sporadic and inconsistent measurements were previously available. In ice, the relaxation rates exhibit an activated temperature dependence with an isotope-independent activation energy. Across 248–273 K, the H2O-to-D2O relaxation rate ratio remains constant at 2.0±0.1. This scaling agrees with Kramers’ theory in the high-friction limit if the moving mass is the proton or deuteron, indicating that dielectric relaxation is governed by a classic proton transfer over an energy barrier rather than molecular reorientation.

Charge and slip-length optimization in lipid-bilayer-coated nanofluidics for enhanced osmotic energy harvesting

Y. Teng; T. Chen; N. Cai; P. Saud; H. Xu et al. 

Tuning Rectification and Gating in Biological Nanopores Through Lumen Charge Mutation

S. F. Mayer; M. F. Mitsioni; M. J. Marcaida; T. Emmerich; A. Radenovic et al. 

β-barrel nanopores are involved in crucial biological processes, from ATP export in mitochondria to antibiotic resistance in Gram-negative bacteria, and represent a promising platform for emerging sequencing technologies. However, in contrast to ion channels, the understanding of the fundamental principles governing ion transport through these nanopores remains in its early stages. In this chapter, we describe the production and mutation of three major biological nanopores-aerolysin, MspA, and α-HL-as well as experimental approaches to elucidate ion transport mechanisms in these biological nanopores. Specifically, we provide tools to characterize two distinct nonlinear phenomena: open-pore rectification and gating, as well as guidelines to tune these phenomena to achieve a certain rectification or gating behavior.

Fixed Aptamer-Functionalized DNA Carriers for Protein Detection with Movable Nanopores

H. Miljkovic; W. Yang; A. Radenovic 

Nanopore sensing has shown great potential; however, one of its major challenges remains the fast and uneven translocation of analytes in free translocation nanopore measurements. We recently introduced Scanning Ion Conductance Spectroscopy (SICS), a technique that enables precise control of translocation speed by immobilizing the analyte on the surface and regulating the speed through controlled capillary movement. This methodology requires a unique preparation of the analyte over conventional free-translocation experiments, as the analyte is captured by and immobilized along a DNA carrier that is attached to a glass surface. This chapter describes the preparation of the essential components required for experiments, including the capillary, buffer, measurement system, and, most importantly, the preparation of DNA carriers that can be functionalized with aptamers.

Concluding remarks: Molecular and ion flows through angstrom-scale channels: bridging theory, simulation, and experiment

A. Radenovic 

This paper summarises the Faraday Discussion on molecular and ion flows through angstrom-sized channels held in Manchester. Photograph of artwork by Felix Kis and Aleksandra Radenovic. Reproduced with permission of the artists and copyright holders.

Optical Sensing with Defects in Two-Dimensional Materials

Scanning ion-conductance spectroscopy and force microscopy of biomolecules

A generalizable codesigned platform for solid-state nanopore sensing beyond the capacitive-noise constraints

N. Cai; W. Guo; Y. Teng; Y. Lou; S-H. Wong et al. 

Solid-state nanopores offer label-free, real-time single-molecule sensing. However, resolving fast biomolecular transport requires high-bandwidth data acquisition while the intrinsic high-frequency noise limits recovery of informative events. Here we present a hardware-software co-designed nanopore sensing platform that combines wafer-scale low-noise device engineering with deep learning-based signal reconstruction. A low-dielectric SU8 coating on silicon nitride nanopores reduces device capacitance to the pF range and suppresses high-frequency noise by up to 5-fold while maintaining facile, controllable and reproducible fabrication. This extends usable acquisition to 40 MHz and enables capture of fast molecular features. Coupled with a reconstruction model trained on synthetic translocation events embedded in experimentally measured noise, the platform recovers transient sublevels while preserving blockage edges and temporal fidelity. Using engineered DNA molecules carrying dumbbell-like barcodes, we resolve nanometer-scale structural spacings on sub-microsecond timescales, and experimentally quantify translocation dynamics within the sub-10 nanometer regime. Dual-channel measurement on a single nanopore device further demonstrates transferability of the platform by showing robust cross-channel signal reconstruction across distinct baseline noise levels. Our approach provides a general route for reliable recovery of fast event features and may enable more information-rich single-molecule sensing across diverse biomolecular targets.

Non-contact direct sensing of material properties of biomolecular condensate using Scanning Ionic Conductance Microscopy

H. Miljkovic; K. Pang; Z. Ayar Dulabi; E. Fatti; A. S. Naidu et al. 

Biomolecular condensates are important regulators of cellular compartmentalization and biochemical processes. Understanding their material properties is critical to elucidate how they control molecular organization and dynamics within cells. However, quantitatively probing these properties remains challenging due to the wide range of length scales, concentrations, and timescales over which condensates operate, as well as the limited force ranges accessible to current nanoscale mechanical mapping methods. We explored the use of a non-contact 3D imaging tool Scanning Ion Conductance Microscopy (SICM) for stiffness measurements of liquid-liquid phase-separated biomolecular condensates. We focus on the Dhh1 protein, which is a regulator of cytoplasmic processing bodies (PBs) membrane-less cytoplasmic condensates that control the storage and degradation of untranslated mRNA. In our study, we investigate the properties of mCherry2- or His-mCherry2- tagged full-length Dhh1 and N- or C-terminus tail-deletion constructs, as well as the catalytically inactive mutant DQAD, under different pH and incubation times. We mapped both spatial and temporal changes in the material properties of the condensates, highlighting the capabilities of the instrument. We found that the removal of either of the two tails led to an increase in condensate stiffness upon shifting the pH from a stress-associated cellular environment (pH 6.5) to physiological conditions (pH 7.5). Additionally, the choice of protein tags led to vastly different results depending on the pH where mCherry2-Dhh1 exhibited a stiffening going from pH 6.0 to 6.5 while the double-tagged His-mCherry2 did not. Our measurements are verified and corroborated by established techniques such as optical tweezer-based fusion assays and fluorescence recovery after photobleaching (FRAP). Furthermore, we were able to track the same biomolecular condensate sample for up to 20 days getting insights on the ageing and evolution of the condensates. Overall, our study demonstrates the applicability of SICM for direct measurement of the material properties of biomolecular condensate.

Nanopore Trap for Label‐Free Fingerprinting of Surface‐modified Single Nanoparticles

N. Cai; T. Chen; Y. Teng; A. S. Naidu; A. Radenovic 

Label‐free characterization of nanoparticle surface functionalization at single‐particle resolution is essential for a wide range of applications. Solid‐state nanopore sensing provides a direct electrical readout that is intrinsically sensitive to the size, surface layer, and interfacial chemistry of single particles in liquid environments. The trapping‐based nanopore sensing regime further enables probing surface‐dependent particle‐pore interactions with extended observation time. Here, a solid‐state nanopore trap‐based fingerprinting method is presented to differentiate single nanoparticles with distinct surface modifications. The method combines a “trap‐release” measurement protocol with a multi‐metric analysis workflow that extracts blockade distributions, sub‐level statistics and frequency‐domain signatures from trapping events, and constructs a unique fingerprint for each particle species. Applied to silica cores (≈25–30 nm) functionalized with APTES, NHS‐PEG 4 ‐Biotin and Tween‐20, the approach generates distinct fingerprints that map to surface charge, coating conformation and configuration heterogeneity. Moreover, in situ detection of surface chemical transformation via specific streptavidin binding is demonstrated, with stoichiometry‐dependent progression of the fingerprints. This platform provides a complementary tool to optical, spectral and ensemble assays for characterizing engineered nanoparticle surfaces and tracking interfacial molecular interactions in solution with label‐free and single‐particle sensitivity.

Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera

N. Ronceray; S. Bennani; M. F. Mitsioni; N. Siegel; M. J. Marcaida et al. 

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally, FLIM uses time-correlated single-photon counting (TCSPC), which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively. Here, we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC, while imaging with a large number of pixels (512 × 512) allowing for the spatial multiplexing of over 3000 molecules. With this approach, we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers, and temporal single-molecule Förster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.

Lumen charge governs gated ion transport in β-barrel nanopores

S. F. Mayer; M. F. Mitsioni; P. Robin; L. van den Heuvel; N. Ronceray et al. 

β-Barrel nanopores are involved in crucial biological processes, from ATP export in mitochondria to bacterial resistance, and represent a promising platform for emerging sequencing technologies. However, in contrast to ion channels, the understanding of the fundamental principles governing ion transport through these nanopores remains largely unexplored. Here we integrate experimental, numerical and theoretical approaches to elucidate ion transport mechanisms in β-barrel nanopores. We identify and characterize two distinct nonlinear phenomena: open-pore rectification and gating. Through extensive mutation analysis of aerolysin nanopores, we demonstrate that open-pore rectification is caused by ionic accumulation driven by the distribution of lumen charges. In addition, we provide converging evidence suggesting that gating is controlled by electric fields dissociating counterions from lumen charges, promoting local structural deformations. Our findings establish a rigorous framework for characterizing and understanding ion transport processes in protein-based nanopores, enabling the design of adaptable nanofluidic biotechnologies. We illustrate this by optimizing an aerolysin mutant for computing applications.

Assessment of neurobehavioural traits under gnotobiotic conditions: an approach for multiple analyses in the same mouse

A. Rutsch; M. Iachizzi; J. Kirundi; J. B. Kantsjö; A. L. Planchette et al. 

The gut-microbiota-brain axis influences neuroinflammation, neural development and behaviour such as sociability, memory and anxiety. To study these traits in vivo, especially during development or disease, it is crucial to analyse them over time and with multiple analyses in the same animal. With a growing understanding of the role of specific bacteria in neurodegenerative disease and behaviour, the demand for gnotobiotic mouse models has increased. However, maintaining stable hygienic conditions during behavioural testing is challenging, as exposure to conventional environments can alter the hygienic status of mice and affect behaviour. We established protocols to perform behavioural tests assessing memory, anxiety, exploration, learning and recognition under axenic conditions using flexible film isolators. Our study compared the behaviour of germ-free mice with mice carrying a defined minimal or moderately diverse microbiota. The results showed no effect of the microbiota on short- and long-term memory or novel object recognition. However, we showed that mice colonised with defined moderately diverse commensal bacteria exhibited more anxiety-like behaviour than germ-free mice. In addition, we showed that microbiota complexity is important, as only mice colonised with moderately diverse microbiota exhibited anxiety-like behaviour, allowing us to disentangle the contribution of specific microbial species or community interactions to this phenotype. This phenotype associated with differences in hippocampal and serum metabolic profiles between colonised and germ-free mice. We propose a novel approach to study rodent behaviour at different physiological and pathological stages in their life without compromising hygiene, thus promoting the refinement and reduction of mice used in experiments.

Deep‐Learning‐Assisted SICM for Enhanced Real‐Time Imaging of Nanoscale Biological Dynamics

Z. Ayar; M. Penedo Garcia; B. Drake; J. Shi; S. M. Leitão et al. 

Scanning Ion Conductance Microscopy (SICM) provides high‐resolution, nanoscale imaging of living cells, but it is generally limited by a slow scan rate, making it challenging to capture dynamic processes in real time. To tackle this challenge, an integrated data acquisition and computational framework is proposed that improves the temporal resolution of SICM by selectively skipping certain scan lines. A partial convolutional neural network (Partial‐CNN) model is developed and trained on SICM images and their corresponding masks to reconstruct the complete images from the undersampled data, ensuring the retention of structural integrity. This approach significantly reduces the image acquisition time (i.e., by 30–63%) without compromising quality, as validated through multiple quantitative metrics. Compared to conventional deep learning methods, the Partial‐CNN demonstrates higher accuracy in reconstructing fine details and maintaining consistent height maps across skipped regions. It is shown that this method provides an increased temporal resolution and retains image fidelity, making it suitable for real‐time dynamic SICM imaging and improving the smart scanning microscopy applications in time‐resolved biological imaging.

Self-driving microscopy detects the onset of protein aggregation and enables intelligent Brillouin imaging

K. A. Ibrahim; C. Cathala; C. Bevilacqua; L. Feletti; R. Prevedel et al. 

The process of protein aggregation, central to neurodegenerative diseases like Huntington’s, is challenging to study due to its unpredictable nature and relatively rapid kinetics. Understanding its biomechanics is crucial for unraveling its role in disease progression and cellular toxicity. Brillouin microscopy offers unique advantages for studying biomechanical properties, yet is limited by slow imaging speed, complicating its use for rapid and dynamic processes like protein aggregation. To overcome these limitations, we developed a self-driving microscope that uses deep learning to predict the onset of aggregation from a single fluorescence image of soluble protein, achieving 91% accuracy. The system triggers optimized multimodal imaging when aggregation is imminent, enabling intelligent Brillouin microscopy of this dynamic biomechanical process. Furthermore, we demonstrate that by detecting mature aggregates in real time using brightfield images and a neural network, Brillouin microscopy can be used to study their biomechanical properties without the need for fluorescence labeling, minimizing phototoxicity and preserving sample health. This autonomous microscopy approach advances the study of aggregation kinetics and biomechanics in living cells, offering a powerful tool for investigating the role of protein misfolding and aggregation in neurodegeneration.

Sum-Frequency Scattering Spectroscopy Reveals the Charging Mechanism and Surface Structure of hBN Nanoflakes in Solution

B. Rehl; N. Ronceray; L. Zhang; A. Rađenović; S. Roke 

A molecular understanding of the interactions between two-dimensional (2D) layered materials and liquids is crucial for nanofluidics, catalysis, and solution-based 2D material processing. Among 2D materials, hexagonal boron nitride (hBN) has a number of outstanding properties, but its interactions with liquids remain poorly characterized. Here, we investigate the interfacial structure of few-layer hBN nanoflakes suspensions in ethanol and ethanol−water mixtures. Electrophoretic light scattering suggests that the nanoflakes are effectively positively charged in ethanol and negatively charged in an ethanol−water mixture. Vibrational sum-frequency scattering spectroscopy reveals the surface structural changes underlying this charge reversal. Signatures of charge transfer of opposite direction are detected on both the flake lattice and in the liquid. The different (partial) charge distributions in ethanol and water explain the apparent charge reversal.