Nanometer Precision Research Articles

Constructing DNAzyme-driven three-dimensional DNA nanomachine-mediated paper-based photoelectrochemical device for ultrasensitive detection of miR-486-5p

As a unique class of dynamic nanostructures, biomimetic DNA walking machines that exhibit geometrical complexity and nanometre precision have gained great success in photoelectrochemical (PEC) bioanalysis. Despite certain achievements, the slow reaction kinetics and low processivity severely restrict the amplification efficiency of the DNA walker-mediated biosensors. Herein, by taking advantage of efficient DNA rolling machines, a three-dimensional (3D) DNA nanomachine-mediated paper-based PEC device for speedy ultrasensitive detection of miR-486–5p was successfully constructed. To achieve it, a novel In2S3/SnS2 sensitized heterojunction was firstly in-situ grown on the Au-modified paper fibers and implemented as the photoanode with effective separation of photogenerated carriers to achieve an enhanced initial photocurrent. Subsequently, the copper hexacyanoferrate(II)-modified CuO nanosphere was introduced as a multifunctional signal regulator via the competitive capture of electron donors and photon energy with the photoelectric layer for efficiently quenching the PEC signal. With the introduction of targets, the DNAzyme-driven DNA nanomachine with editable motion modes was gradually activated and it could continuously cleave the tracks DNA labeled quenching probes, finally achieving the recovery of PEC signal. As a proof of concept, the elaborated paper-based PEC device presented a wide linear range from 0.1 fM to 100 pM and a detection limit of 35 aM for miR-486–5p bioassay. This work provides an innovative insight to the exploitation of DNA nanobiotechnology and nucleic acid signal amplification strategy.

The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices.

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy. However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts - and knowing how to avoid them - takes on special urgency.We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a 'before-and-after' experiment. We applied super-resolution shadow imaging to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters.While the ECS volume fraction and micro-anatomical organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animals in vivo where the ECS typically becomes nearly depleted.Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphological preservation of cells and tissues.Significance StatementChemical fixation of biological samples using PFA is a standard step routinely performed in neuroscience labs. However, it is known to alter various anatomical parameters ranging from protein distribution to cell morphology, potentially affecting our interpretation of anatomical data. With the increasing use of super-resolution microscopy, understanding the extent and nature of fixation artifacts is an urgent concern.Here, we use live STED microscopy to monitor in real time the impact of PFA on the microanatomy of organotypic hippocampal brain slices. Our results demonstrate that while PFA has little impact on the extracellular space and astrocytes, it compromises cell membranes and dendritic structures. Our study provides a strategy for a direct characterization of fixation artifacts at the nanoscale, facilitating the optimization of fixation protocols.

Optimizing self-interference digital holography for single-molecule localization.

Self-interference digital holography (SIDH) can image incoherently emitting objects over large axial ranges from three two-dimensional images. By combining SIDH with single-molecule localization microscopy (SMLM), incoherently emitting objects can be localized with nanometer precision over a wide axial range without mechanical refocusing. However, background light substantially degrades the performance of SIDH due to the relatively large size of the hologram. To optimize the performance of SIDH, we performed simulations to study the optimal hologram radius (Rh) for different levels of background photons. The results show that by reducing the size of the hologram, we can achieve a localization precision of better than 60nm laterally and 80 nm axially over a 10 µm axial range under the conditions of low signal level (6000 photons) with 10 photons/pixel of background noise. We then performed experiments to demonstrate our optimized SIDH system. The results show that point sources emitting as few as 2120 photons can be successfully detected. We further demonstrated that we can successfully reconstruct point-like sources emitting 4200 photons over a 10 µm axial range by light-sheet SIDH.

Positional control of DNA origami based gold dimer hybrid nanostructures on pre-structured surfaces

This study explores important parameters for achieving a high-level positional control of DNA-nanoparticle hybrid structures by drop-casting onto a pre-structured silicon surface, in which the active adsorption sites were defined using electron beam lithography. By confining the adsorption sites to the scale of the DNA origami, we create multi-dimensional patterns and study the effect of diffusion and hybrid nanostructure concentration in the liquid on site occupation. We also propose a physical diffusion model that highlights the importance of surface diffusion in facilitating the adsorption of hybrid nanostructure onto active sites, particularly for two and one-dimensional adsorption sites. Our study shows prominent results of the hybrid nanostructure’s selective adsorption, indicating high adsorption efficiency and precise control over the position, as well as the spatial orientation. We anticipate similar results in related systems, both in terms of different surfaces and similar DNA structures. Overall, our findings offer promising prospects for the development of large-scale nanoarrays on micrometer-scale surfaces with nanometer precision and orientation control.

Quantitative Determination of Surface Morphology of Red Blood Cell

In this study, the determination of the surface morphology of red blood cell (RBC) from interferogram image obtained by quantitative phase imaging (QPI) method is presented. QPI, is an optical measurement method frequently used in recent years, allows to obtain quantitative data for different samples (cell, thin film surface, etc.). Many measurement setups at the micrometer level and with nanometer precision have been designed for quantitative surface determination. Among these, white light diffraction phase microscopy (WDPM) is a design that combines the advantages of off-axis holography-specific speed and phase sensitivity associated with common path interferometry. Interferogram image of RBCs have been formed by the WDPM setup. Analysis of this image has been carried out by Fourier transform. As a result of this analysis, three-dimensional (3D), dynamic (observable from all angles) and height-known profiles of RBCs have been created. From the height profiles, the parameters related to the morphology of RBCs as the projected surface area (PSA), diameter (D), mean corpuscular volume (MCV) and total surface area occupied by the cell (SA), have been determined quantitatively. In addition, two-dimensional images, obtained by examining blood samples with light microscopy and scanning electron microscopy (SEM), have been compared with the data achieved by WDPM. The advantages and disadvantages of WDPM and light microscopy and SEM, which are commonly used in biomedical measurements, are discussed through the results. In this way, it was possible to see the difference between QPI and traditional methods used to imaging the cell surface.

Remodeling of the focal adhesion complex by hydrogen-peroxide-induced senescence

Cellular senescence is a phenotype characterized by cessation of cell division, which can be caused by exhaustive replication or environmental stress. It is involved in age-related pathophysiological conditions and affects both the cellular cytoskeleton and the prime cellular mechanosensors, focal adhesion complexes. While the size of focal adhesions increases during senescence, it is unknown if and how this is accompanied by a remodeling of the internal focal adhesion structure. Our study uses metal-induced energy transfer to study the axial dimension of focal adhesion proteins from oxidative-stress-induced senescent cells with nanometer precision, and compares these to unstressed cells. We influenced cytoskeletal tension and the functioning of mechanosensitive ion channels using drugs and studied the combined effect of senescence and drug intervention on the focal adhesion structure. We found that H2O2-induced restructuring of the focal adhesion complex indicates a loss of tension and altered talin complexation. Mass spectroscopy-based proteomics confirmed the differential regulation of several cytoskeletal proteins induced by H2O2 treatment.

Local Photochemical Nanoscopy of Hot-Carrier-Driven Catalytic Reactions Using Plasmonic Nanosystems.

Nanoscale investigation of the reactivity of photocatalytic systems is crucial for their fundamental understanding and improving their design and applicability. Here, we present a photochemical nanoscopy technique that unlocks the local spatial detection of molecular products during plasmonic hot-carrier-driven photocatalytic reactions with nanometric precision. By applying the methodology to Au/TiO2 plasmonic photocatalysts, we experimentally and theoretically determined that smaller and denser Au nanoparticle arrays present lower optical contribution with quantum efficiency in hot-hole-driven photocatalysis closely related to the population heterogeneity. As expected, the highest quantum yield from a redox probe oxidation is achieved at the plasmon peak. Investigating a single plasmonic nanodiode, we unravel the areas where oxidation and reduction products are evolved with subwavelength resolution (∼200 nm), illustrating the bipolar behavior of such nanosystems. These results open the way to quantitative investigations at the nanoscale to evaluate the photocatalytic reactivity of low-dimensional materials in a variety of chemical reactions.

NANOCRINOLOGY.

We conceptualize and define nanocrinology as the science that studies the nanometric and subnanometric precision that operates in diagnostic and therapeutic endocrinology. It includes advanced generation assays, which can detect low concentrations of hormones, and modern drug delivery systems that allow more efficient delivery of endocrinotropic agents. Nanocrinology is a rapidly growing field of endocrinology, and we call for greater research and adoption of this science.

Combining deep learning approaches and point spread function engineering for simultaneous 3D position and 3D orientation measurements of fluorescent single molecules

Point Spread Function (PSF) engineering is an effective method to increase the sensitivity of single-molecule fluorescence images to specific parameters. Classical phase mask optimization approaches have enabled the creation of new PSFs that can achieve, for example, localization precision of a few nanometers axially over a capture range of several microns with bright emitters. However, for complex high-dimensional optimization problems, classical approaches are difficult to implement and can be very time-consuming for computation. The advent of deep learning methods and their application to single-molecule imaging has provided a way to solve these problems. Here, we propose to combine PSF engineering and deep learning approaches to obtain both an optimized phase mask and a neural network structure to obtain the 3D position and 3D orientation of fixed fluorescent molecules. Our approach allows us to obtain an axial localization precision around 30 nanometers, as well as an orientation precision around 5 degrees for orientations and positions over a one micron depth range for a signal-to-noise ratio consistent with what is typical in single-molecule cellular imaging experiments.

DNA origami technology for biomedical applications: Challenges and opportunities

AbstractDNA origami, a promising branch of structural DNA technology, refers to the technique of folding a single‐stranded DNA scaffold into well‐defined nanostructures. In recent years, DNA origami nanostructures have shown considerable promise in a variety of biomedical applications, owing to their biodegradability, unique programmability, and addressability. Despite their popularity, the biomedical application of DNA origami techniques, which exploits their unique programmability and addressability, is rare in previous studies. Most recently, mounting evidence has demonstrated the robustness of DNA origami nanostructures in the spatial organization of functional components at the nanoscale in the biomedical field. These examples provide typical paradigms to fully realize the potential of DNA origami techniques by taking advantage of their unique programmability and addressability. This minireview summarizes the recent advancements of DNA origami techniques in biosensing, biocatalysis, and drug delivery, and the representative examples using DNA origami nanostructures for the spatial organization of functional molecules with nanometric precision are highlighted. We further discuss the possible limitations and challenges for in vivo applications, including stability issues and potential immunogenicity, and finally, propose some strategies to overcome these obstacles to fully realize the potential of DNA origami techniques in biomedical applications.

Self-Limiting Sub-5 nm Nanodiamonds by Geochemistry-Inspired Synthesis.

Controlling diamond structures with nanometer precision is fundamentally challenging owing to their extreme and far-from-equilibrium synthetic conditions. State-of-the-art techniques, including detonation, chemical vapor deposition, mechanical grinding, and high-pressure-high-temperature synthesis, yield nanodiamond particles with a broad distribution of sizes. Despite many efforts, the direct synthesis of nanodiamonds with precisely controlled diameters remains elusive. Here the geochemistry-inspired synthesis of sub-5 nm nanodiamonds with sub-nanometer size deviation is described. High-pressure-high-temperature treatment of uniform iron carbide nanoparticles embedded in iron oxide matrices yields nanodiamonds with tunable diameters down to 2.13 and 0.22nm standard deviation. A self-limiting, redox-driven, and diffusion-controlled solid-state reaction mechanism is proposed and supported by in situ X-ray diffraction, ex situ characterizations, and computational modeling. This work provides a unique mechanism for the precise control of nanostructured diamonds under extreme conditions and paves the road for the full realization of their potential in emerging technologies.

Correlative light-electron microscopy using small gold nanoparticles as single probes

Correlative light-electron microscopy (CLEM) requires the availability of robust probes which are visible both in light and electron microscopy. Here we demonstrate a CLEM approach using small gold nanoparticles as a single probe. Individual gold nanoparticles bound to the epidermal growth factor protein were located with nanometric precision background-free in human cancer cells by light microscopy using resonant four-wave mixing (FWM), and were correlatively mapped with high accuracy to the corresponding transmission electron microscopy images. We used nanoparticles of 10 nm and 5 nm radius, and show a correlation accuracy below 60 nm over an area larger than 10 µm size, without the need for additional fiducial markers. Correlation accuracy was improved to below 40 nm by reducing systematic errors, while the localisation precision is below 10 nm. Polarisation-resolved FWM correlates with nanoparticle shapes, promising for multiplexing by shape recognition in future applications. Owing to the photostability of gold nanoparticles and the applicability of FWM microscopy to living cells, FWM-CLEM opens up a powerful alternative to fluorescence-based methods.

A Dynamic Control Center Based on a DNA Reaction Network for Programmable Building of DNA Nanostructures.

DNA-based nanostructures allow for complex self-assembly with nanometer precision through the specificity of Watson-Crick base pairing, but network behavior-directed control of the kinetic process is less studied. Here we show how the DNA reaction network (DRN), which has emerged as a reliable and programmable way to implement artificial network dynamics, can be built as the control center of programmable nanostructures, allowing spatiotemporal control over the dynamic behavior of DNA nanotubes. We chose a common network motif in biological control systems, the feed-forward loop, as the model network and demonstrated that dynamic behaviors, such as self-tuning control and multilayer hierarchical assembly, could be programmed by constructing an inhibition network and an excitation network, separately, in buffer solution and inside protocells.

Real-space observation of a two-dimensional electron gas at semiconductor heterointerfaces.

Mobile charge carriers are essential components in high-performance, nano-engineered semiconductor devices. Employing charge carriers confined to heterointerfaces, the so-called two-dimensional electron gas, is essential for improving device performance. The real-space visualization of a two-dimensional electron gas at the nanometre scale is desirable. However, it is challenging to accomplish by means of electron microscopy due to an unavoidable strong diffraction contrast formation at the heterointerfaces. We performed direct, nanoscale electric field imaging across a GaN-based semiconductor heterointerface using differential phase contrast scanning transmission electron microscopy by suppressing diffraction contrasts. For both nearly the lattice-matched GaN/Al0.81In0.19N interface and pseudomorphic GaN/Al0.88In0.12N interface, the extracted quantitative electric field profiles show excellent agreement with profiles predicted using Poisson simulation. Furthermore, we used the electric field profiles to quantify the density and distribution of the two-dimensional electron gas across the heterointerfaces with nanometre precision. This study is expected to guide the real-space characterization of local charge carrier density and distribution in semiconductor devices.

Constructing hierarchical structures of Pd catalysts to realize reaction pathway regulation of furfural hydroconversion

The technique of controlling nanoscale features precisely has been exploited to develop and improve bifunctional catalysts, which are important in the hydroconversion of furfural to high-value chemicals. Such bifunctional hydroconversion catalysts consist of metal sites and acid sites, and the intimacy extent of both active sites dictates the catalytic performance. In-depth exploration of the intimacy extent has been long limited by the lack of material synthesis and characterization method with nanometer precision. Herein, hierarchical catalytic structures with the spatial separation of Pd and acid sites were constructed by the multistep deposition strategy, wherein Pd was confined on defective CeO2 nanoclusters, which were dispersed on high-surface-area SiO2 with the strong acidity. The intimacy extent of Pd and acid sites in hierarchical catalytic structures of Pd/CeO2/SiO2 can be modulated by adjusting the CeO2 content, which leads to the aqueous-phase hydrogenation of furfural following a unique treble-site mechanism. The perpendicular adsorption of furfural on enriched oxygen vacancies of Pd/CeO2 contributes to the selective hydrogenation of the carbonyl group, and then the formed furfuryl alcohol undergoes the acid-catalyzed rearrangement of furan rings on SiO2 through the cooperative effect of acid sites and hydrogen spillover to give cyclopentanone. Meanwhile, the asynchronous catalysis effectively hinders the furan ring hydrogenation to tetrahydrofurfuryl alcohol by spatially separating Pd from the SiO2 surface. The superior catalytic performance is obtained on Pd/CeO2/SiO2-10 with a furfural conversion of 93% and cyclopentanone selectivity of 84%. We anticipate that the strategy of constructing hierarchical catalytic structures demonstrated here to spatially organize different active sites at the nanoscale will boost the further development of multifunctional catalysts.

Photoactivatable BODIPYs for Live-Cell PALM.

Photoactivated localization microscopy (PALM) relies on fluorescence photoactivation and single-molecule localization to overcome optical diffraction and reconstruct images of biological samples with spatial resolution at the nanoscale. The implementation of this subdiffraction imaging method, however, requires fluorescent probes with photochemical and photophysical properties specifically engineered to enable the localization of single photoactivated molecules with nanometer precision. The synthetic versatility and outstanding photophysical properties of the borondipyrromethene (BODIPY) chromophore are ideally suited to satisfy these stringent requirements. Specifically, synthetic manipulations of the BODIPY scaffold can be invoked to install photolabile functional groups and photoactivate fluorescence under photochemical control. Additionally, targeting ligands can be incorporated in the resulting photoactivatable fluorophores (PAFs) to label selected subcellular components in live cells. Indeed, photoactivatable BODIPYs have already allowed the sub-diffraction imaging of diverse cellular substructures in live cells using PALM and can evolve into invaluable analytical probes for bioimaging applications.

Vertical growth dynamics of biofilms

During the biofilm life cycle, bacteria attach to a surface and then reproduce, forming crowded, growing communities. Many theoretical models of biofilm growth dynamics have been proposed; however, difficulties in accurately measuring biofilm height across relevant time and length scales have prevented testing these models, or their biophysical underpinnings, empirically. Using white light interferometry, we measure the heights of microbial colonies with nanometer precision from inoculation to their final equilibrium height, producing a detailed empirical characterization of vertical growth dynamics. We propose a heuristic model for vertical growth dynamics based on basic biophysical processes inside a biofilm: diffusion and consumption of nutrients and growth and decay of the colony. This model captures the vertical growth dynamics from short to long time scales (10 min to 14 d) of diverse microorganisms, including bacteria and fungi.

Axial profiling of interferometric scattering enables an accurate determination of nanoparticle size.

Interferometric scattering (iSCAT) microscopy has undergone significant development in recent years. It is a promising technique for imaging and tracking nanoscopic label-free objects with nanometer localization precision. The current iSCAT-based photometry technique allows quantitative estimation for the size of a nanoparticle by measuring iSCAT contrast and has been successfully applied to nano-objects smaller than the Rayleigh scattering limit. Here we provide an alternative method that overcomes such size limitations. We take into account the axial variation of iSCAT contrast and utilize a vectorial point spread function model to uncover the position of a scattering dipole and, consequently, the size of the scatterer, which is not limited to the Rayleigh limit. We found that our technique accurately measures the size of spherical dielectric nanoparticles in a purely optical and non-contact way. We also tested fluorescent nanodiamonds (fND) and obtained a reasonable estimate for the size of fND particles. Together with fluorescence measurement from fND, we observed a correlation between the fluorescent signal and the size of fND. Our results showed that the axial pattern of iSCAT contrast provides sufficient information for the size of spherical particles. Our method enables us to measure the size of nanoparticles from tens of nanometers and beyond the Rayleigh limit with nanometer precision, making a versatile all-optical nanometric technique.

Self-supervised machine learning pushes the sensitivity limit in label-free detection of single proteins below 10 kDa

Interferometric scattering (iSCAT) microscopy is a label-free optical method capable of detecting single proteins, localizing their binding positions with nanometer precision, and measuring their mass. In the ideal case, iSCAT is limited by shot noise such that collection of more photons should extend its detection sensitivity to biomolecules of arbitrarily low mass. However, a number of technical noise sources combined with speckle-like background fluctuations have restricted the detection limit in iSCAT. Here, we show that an unsupervised machine learning isolation forest algorithm for anomaly detection pushes the mass sensitivity limit by a factor of 4 to below 10 kDa. We implement this scheme both with a user-defined feature matrix and a self-supervised FastDVDNet and validate our results with correlative fluorescence images recorded in total internal reflection mode. Our work opens the door to optical investigations of small traces of biomolecules and disease markers such as α-synuclein, chemokines and cytokines.

In-situ atomic level observation of the strain response of graphene lattice

Strain is inevitable in two-dimensional (2D) materials, regardless of whether the film is suspended or supported. However, the direct measurement of strain response at the atomic scale is challenging due to the difficulties of maintaining both flexibility and mechanical stability at low temperature under UHV conditions. In this work, we have implemented a compact nanoindentation system with a size of sim 160 mm^2times 5.2 mm in a scanning tunneling microscope (STM) sample holder, which enables the reversible control of strain and gate electric field. A combination of gearbox and piezoelectric actuator allowed us to modulate the depth of the indentation continuously with nanometer precision. The 2D materials were transferred onto the polyimide film. Pd clamp was used to enhance the strain transfer from the polyimide from to the 2D layers. Using this unique technique, strain response of graphene lattice were observed at atomic precision. In the relaxed graphene, strain is induced mainly by local curvature. However, in the strained graphene with tented structure, the lattice parameters become more sensitive to the indentor height change and stretching strain is increased additionally. Moreover, the gate controllability is confirmed by measuring the dependence of the STM tip height on gate voltage.