Spatially multiplexed single-molecule translocations through a nanopore at controlled speeds.

In current nanopore-based label-free single-molecule sensing technologies, stochastic processes influence the selection of translocating molecule, translocation rate and translocation velocity. As a result, single-molecule translocations are challenging to control both spatially and temporally. Here we present a method using a glass nanopore mounted on a three-dimensional nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at a controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio by two orders of magnitude compared with free translocations. We demonstrate the method's versatility by assessing DNA-protein complexes, DNA rulers and DNA gaps, achieving down to single-nucleotide gap detection.

Spectral cross-cumulants for multicolor super-resolved SOFI imaging.

Super-resolution optical fluctuation imaging provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution and the sampling can be increased up to n-fold in all spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increases the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and experiments in fixed and live cells. Based on an eigenvalue/vector analysis, we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property.

Parameter-free image resolution estimation based on decorrelation analysis.

Super-resolution microscopy opened diverse new avenues of research by overcoming the resolution limit imposed by diffraction. Exploitation of the fluorescent emission of individual fluorophores made it possible to reveal structures beyond the diffraction limit. To accurately determine the resolution achieved during imaging is challenging with existing metrics. Here, we propose a method for assessing the resolution of individual super-resolved images based on image partial phase autocorrelation. The algorithm is model-free and does not require any user-defined parameters. We demonstrate its performance on a wide variety of imaging modalities, including diffraction-limited techniques. Finally, we show how our method can be used to optimize image acquisition and post-processing in super-resolution microscopy.

Electrochemical Reaction in Single Layer MoS2: Nanopores Opened Atom by Atom.

Ultrathin nanopore membranes based on 2D materials have demonstrated ultimate resolution toward DNA sequencing. Among them, molybdenum disulfide (MoS2) shows long-term stability as well as superior sensitivity enabling high throughput performance. The traditional method of fabricating nanopores with nanometer precision is based on the use of focused electron beams in transmission electron microscope (TEM). This nanopore fabrication process is time-consuming, expensive, not scalable, and hard to control below 1 nm. Here, we exploited the electrochemical activity of MoS2 and developed a convenient and scalable method to controllably make nanopores in single-layer MoS2 with subnanometer precision using electrochemical reaction (ECR). The electrochemical reaction on the surface of single-layer MoS2 is initiated at the location of defects or single atom vacancy, followed by the successive removals of individual atoms or unit cells from single-layer MoS2 lattice and finally formation of a nanopore. Step-like features in the ionic current through the growing nanopore provide direct feedback on the nanopore size inferred from a widely used conductance vs pore size model. Furthermore, DNA translocations can be detected in situ when as-fabricated MoS2 nanopores are used. The atomic resolution and accessibility of this approach paves the way for mass production of nanopores in 2D membranes for potential solid-state nanopore sequencing.

The emergence of nanopores in next-generation sequencing.

Next-generation sequencing methods based on nanopore technology have recently gained considerable attention, mainly because they promise affordable and fast genome sequencing by providing long read lengths (5 kbp) and do not require additional DNA amplification or enzymatic incorporation of modified nucleotides. This permits health care providers and research facilities to decode a genome within hours for less than $1000. This review summarizes past, present, and future DNA sequencing techniques, which are realized by nanopore approaches such as those pursued by Oxford Nanopore Technologies.

Probing the size of proteins with glass nanopores.

Single molecule studies using nanopores have gained attention due to the ability to sense single molecules in aqueous solution without the need to label them. In this study, short DNA molecules and proteins were detected with glass nanopores, whose sensitivity was enhanced by electron reshaping which decreased the nanopore diameter and created geometries with a reduced sensing length. Further, proteins having molecular weights (MW) ranging from 12 kDa to 480 kDa were detected, which showed that their corresponding current peak amplitude changes according to their MW. In the case of the 12 kDa ComEA protein, its DNA-binding properties to an 800 bp long DNA molecule was investigated. Moreover, the influence of the pH on the charge of the protein was demonstrated by showing a change in the translocation direction. This work emphasizes the wide spectrum of detectable molecules using nanopores from glass nanocapillaries, which stand out because of their inexpensive, lithography-free, and rapid manufacturing process.

Challenges in quantitative single molecule localization microscopy.

Single molecule localization microscopy (SMLM), which can provide up to an order of magnitude improvement in spatial resolution over conventional fluorescence microscopy, has the potential to be a highly useful tool for quantitative biological experiments. It has already been used for this purpose in varied fields in biology, ranging from molecular biology to neuroscience. In this review article, we briefly review the applications of SMLM in quantitative biology, and also the challenges involved and some of the solutions that have been proposed. Due to its advantages in labeling specificity and the relatively low overcounting caused by photoblinking when photo-activable fluorescent proteins (PA-FPs) are used as labels, we focus specifically on Photo-Activated Localization Microscopy (PALM), even though the ideas presented might be applicable to SMLM in general. Also, we focus on the following three quantitative measurements: single molecule counting, analysis of protein spatial distribution heterogeneity and co-localization analysis.

Progress in quantitative single-molecule localization microscopy.

With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM).

Detecting the translocation of DNA through a nanopore using graphene nanoribbons.

Solid-state nanopores can act as single-molecule sensors and could potentially be used to rapidly sequence DNA molecules. However, nanopores are typically fabricated in insulating membranes that are as thick as 15 bases, which makes it difficult for the devices to read individual bases. Graphene is only 0.335 nm thick (equivalent to the spacing between two bases in a DNA chain) and could therefore provide a suitable membrane for sequencing applications. Here, we show that a solid-state nanopore can be integrated with a graphene nanoribbon transistor to create a sensor for DNA translocation. As DNA molecules move through the pore, the device can simultaneously measure drops in ionic current and changes in local voltage in the transistor, which can both be used to detect the molecules. We examine the correlation between these two signals and use the ionic current measurements as a real-time control of the graphene-based sensing device.

Enhancement of second harmonic signal in nanofabricated cones.

Geometrical effects in optical nanostructures on nanoscale can lead to interesting phenomena such as inhibition of spontaneous emission,1,2 high-reflecting omnidirectional mirrors, structures that exhibit low-loss-waveguiding,3 and light confinement.4,5 Here, we demonstrate a similar concept of exploiting the geometrical effects on nanoscale through precisely fabricating lithium niobate (LiNbO3) nanocones arrays devices. We show a strong second harmonic generation (SHG) enhancement, shape and arrangement dependent, up to 4 times bigger than the bulk one. These devices allow below diffraction limited observation, being perfect platforms for single molecule fluorescence microscopy6 or single cell endoscopy.7 Nanocones create a confined illumination volume, devoid from blinking and bleaching, which can excite molecules in nanocones proximity. Illumination volume can be increased by combining the SH enhancement effect with plasmon resonances, excited thanks to a gold plasmonic shell deposited around the nanostructures. This results in a local further enhancement of the SH signal up to 20 times. The global SH enhancement can be rationally designed and tuned through the means of simulations.

Detection of RNAP-DNA complexes using solid state nanopores.

Transcription is the first step in gene expression where DNA is copied into RNA. It is extensively studied at the bulk level especially the regulation mechanism, which in cancerous cells is impaired. We were interested in studying E. coli RNAP enzyme at the single-molecule level for its functional as well as molecular motor properties. With nanopore sensing, we were able to observe RNA polymerase-DNA complexes translocate through nanopores and able to distinguish between individual complexes and bare RNA polymerase. We were also able to observe orientation of RNA polymerase in the nanopore whether flow or electric field predominates. The complexity of the signals from the protein-DNA complexes experiment motivated us to develop level detection software. This software is based on a change detection method called the CUSUM algorithm. OpenNanpore software was designed to analyze in details current blockages in nanopore signals with very little prior knowledge on the signal. With this work one can separate events according to their number of levels and study those sub-populations separately.

Controllable shrinking and shaping of glass nanocapillaries under electron irradiation.

The ability to reshape nanopores and observe their shrinkage under an electron microscope is a powerful and novel technique. It increases the sensitivity of the resistive pulse sensing and enables to detect very short and small molecules. However, this has not yet been shown for glass nanocapillaries. In contrast to their solid-state nanopore counterparts, nanocapillaries are cheap, easily fabricated and in the production do not necessitate clean room facilities. We show for the first time that quartz nanocapillaries can be shrunken under a scanning electron microscope beam. Since the shrinking is caused by the thermal heating of the electrons, increasing the beam current increases the shrink rate. Higher acceleration voltage on the contrary increases the electron penetration depth and reduces the electron density causing slower shrinkage. This allows us to fine control the shrink rate and to stop the shrinking process at any desired diameter. We show that a shrunken nanocapillary detects DNA translocation with six times higher signal amplitudes than an unmodified nanocapillary. This will open a new path to detect small and short molecules such as proteins or RNA with nanocapillaries.

Fast and automatic processing of multi-level events in nanopore translocation experiments.

We have developed a method to analyze in detail, translocation events providing a novel and flexible tool for data analysis of nanopore experiments. Our program, called OpenNanopore, is based on the cumulative sums algorithm (CUSUM algorithm). This algorithm is an abrupt change detection algorithm that provides fitting of current blockages, allowing the user to easily identify the different levels in each event. Our method detects events using adaptive thresholds that adapt to low-frequency variations in the baseline. After event identification, our method uses the CUSUM algorithm to fit the levels inside every event and automatically extracts their time and amplitude information. This facilitates the statistical analysis of an event population with a given number of levels. The obtained information improves the interpretation of interactions between the molecule and nanopore. Since our program does not require any prior information about the analyzed molecules, novel molecule-nanopore interactions can be characterized. In addition our program is very fast and stable. With the progress in fabrication and control of the translocation speed, in the near future, our program could be useful in identification of the different bases of DNA.

Nanopore detection of single molecule RNAP-DNA transcription complex.

In the past decade, a number of single-molecule methods have been developed with the aim of investigating single protein and nucleic acid interactions. For the first time we use solid-state nanopore sensing to detect a single E. coli RNAP-DNA transcription complex and single E. coli RNAP enzyme. On the basis of their specific conductance translocation signature, we can discriminate and identify between those two types of molecular translocations and translocations of bare DNA. This opens up a new perspectives for investigating transcription processes at the single-molecule level.

Nonlinear optical response in single alkaline niobate nanowires.

We have synthesized and characterized three types of perovskite alkaline niobate nanowires: NaNbO(3), KNbO(3), and LiNbO(3) (XNbO(3)). All three types of nanowires exhibit strong nonlinear response. Confocal imaging has been employed to quantitatively compare the efficiency of synthesized nanowires to generate second harmonic signal and to show that LiNbO(3) nanowires exhibit the strongest nonlinear response. We also investigated the polarization response of the second harmonic generation (SHG) signal in all three types of alkaline nanowires for the two geometries tractable by our optical trapping setup. The SHG signal is highly influenced by the nanowire crystallinity and experimental geometry. We also demonstrate for the first time wave-guiding of SHG signal in all three types of alkaline niobate nanowires. By carefully examining nonlinear properties of (XNbO(3)) nanowires we suggest which type of wires are best suited for the given application.

Single-layer MoS2 transistors.

Two-dimensional materials are attractive for use in next-generation nanoelectronic devices because, compared to one-dimensional materials, it is relatively easy to fabricate complex structures from them. The most widely studied two-dimensional material is graphene, both because of its rich physics and its high mobility. However, pristine graphene does not have a bandgap, a property that is essential for many applications, including transistors. Engineering a graphene bandgap increases fabrication complexity and either reduces mobilities to the level of strained silicon films or requires high voltages. Although single layers of MoS(2) have a large intrinsic bandgap of 1.8 eV (ref. 16), previously reported mobilities in the 0.5-3 cm(2) V(-1) s(-1) range are too low for practical devices. Here, we use a halfnium oxide gate dielectric to demonstrate a room-temperature single-layer MoS(2) mobility of at least 200 cm(2) V(-1) s(-1), similar to that of graphene nanoribbons, and demonstrate transistors with room-temperature current on/off ratios of 1 × 10(8) and ultralow standby power dissipation. Because monolayer MoS(2) has a direct bandgap, it can be used to construct interband tunnel FETs, which offer lower power consumption than classical transistors. Monolayer MoS(2) could also complement graphene in applications that require thin transparent semiconductors, such as optoelectronics and energy harvesting.