True Resolution Research Articles

Lateral Resolution of a Commercial Optical Coherence Tomography Instrument.

PurposeThe lateral resolution of an optical coherence tomography (OCT) instrument was considered to be equal to the illumination spot size on the retina. To evaluate the potential lateral resolution of the Spectralis OCT, an instrument calculated to have a 14 µm resolution.MethodsThe lateral point spread function (PSF) was evaluated using diamond abrasive powder 0 to 1 µm in diameter in silicone elastomer and a validated target with 800 nm FeO particles in urethane. The amplitude transfer function was calculated from human OCT images. Finally, resolution was measured using the 1951 USAF target.ResultsMeasurement of the lateral PSF from 1215 diamond particle images yielded a full-width half maximum (FWHM) to be 5.11 µm and for 732 FeO particles, 4.9 µm. From the amplitude transfer function, the FWHM of the diffraction limited PSF was calculated to be 5.0 µm. The USAF target imaging showed a lateral resolution of 4.6 µm.ConclusionsAlthough a calculation of the spot size of the illumination beam was reported in the past as the lateral resolution of the OCT instrument, the actual lateral resolution is better by a factor of at least 2.5 times. The clinically used A-scan spacing was derived from the calculated, and not the true resolution, and results in under sampling. This set of findings likely apply to all commercial clinical instruments.Translational RelevanceThe scan density parameters of past and present commercial OCT instruments were based on earlier translational concepts, which now appear to have been incorrect.

Fluorescence nanoscopy at the sub-10nm scale.

Fluorescence nanoscopy represented a breakthrough for the life sciences as it delivers 20-30nm resolution using far-field fluorescence microscopes. This resolution limit is not fundamental but imposed by the limited photostability of fluorophores under ambient conditions. This has motivated the development of a second generation of fluorescence nanoscopy methods that aim to deliver sub-10nm resolution, reaching the typical size of structural proteins and thus providing true molecular resolution. In this review, we present common fundamental aspects of these nanoscopies, discuss the key experimental factors that are necessary to fully exploit their capabilities, and discuss their current and future challenges.

Kinematics and structure of Ionized gas in the UCHII regions of W33 main

Abstract High mass proto-stars create Ultra-Compact H ii regions (UCHII) at the stage of evolution when most of the accretion is finished but the star is still heavily embedded in molecular material. The morphologies of UCHII regions reflect the interactions of stellar winds, stellar motions, and density structure in the molecular cloud; they are complex and it has been very difficult to interpret them. We here present data obtained with TEXES on the NASA IRTF of the [Nei,ii] emission line in the proto-cluster of young OB stars in W33 Main. The data cube has a spatial resolution of ∼1.4 arcsec and true velocity resolution ∼5 km s-1; with Aλ ∼ 0.02AV it is relatively unaffected by extinction. We have run 3D hydrodynamic and line profile simulations, using PLUTO and RADMC-3D, of the gas structures created by multiple windy stars moving relative to each other through the ambient cloud. By iterative comparison of the data cube and the simulations, we arrive at models that reproduce the different morphology and kinematic structure of each UCHII region in W33 Main. The results indicate that each sub-source probably holds multiple exciting stars, permitting an improved view of the stellar population, and finds the stellar trajectories, which may determine the dynamical development of the proto-cluster.

Bringing Electrochemical Three-Dimensional Printing to the Nanoscale

Nanoscale 3D printing is attracting attention as an alternative manufacturing technique for a variety of applications from electronics and nanooptics to sensing, nanorobotics, and energy storage. The constantly shrinking critical dimension in state-of-the-art technologies requires fabrication of complex conductive structures with nanometer resolution. Electrochemical techniques are capable of producing impurity-free metallic conductors with superb electrical and mechanical properties, however, true nanoscale resolution (<100 nm) remained unattainable. Here, we set new a benchmark in electrochemical 3D printing. By employing nozzles with dimensions as small as 1 nm, we demonstrate layer-by-layer manufacturing of 25 nm diameter voxels. Full control of the printing process allows adjustment of the feature size on-the-fly, printing tilted, and overhanging structures. On the basis of experimental evidence, we estimate the limits of electrochemical 3D printing and discuss the origins of this new resolution frontier.

Advanced-sectioned images obtained by microsectioning of the entire male body.

Realistic two-dimensional (2D) and three-dimensional (3D) applications for anatomical studies are being developed from true-colored sectioned images. We generated advanced-sectioned images of the entire male body and verified that anatomical structures of both normal and abnormal shapes could be visualized in them. The cadaver was serially sectioned at constant intervals using a cryomacrotome. The sectioned surfaces were photographed using a digital camera to generate horizontal advanced-sectioned images in which normal and abnormal structures were classified. Advanced-sectioned images of the entire male body were generated. The image resolution was 3.3 × 3.3 fold better than that of the first sectioned images obtained in 2002. In the advanced-sectioned images, normal and abnormal structures ranging from microscopic (≥0.06 mm × 0.06 mm; pixel size) to macroscopic (≤473.1 mm × 202 mm; body size) could be identified. Furthermore, the real shapes and actual sites of lung cancer and lymph node enlargement were ascertained in them. Such images will be useful because of their true color and high resolution in digital 2D and 3D applications for gross anatomy and clinical anatomy. In future, we plan to generate new advanced-sectioned images of abnormal cadavers with different diseases for clinical anatomy studies.

Single-molecule localization microscopy as an emerging tool to probe multiscale food structures

Optical microscopy is an indispensable tool to characterize the microstructure of foods at ambient conditions. Depending on both the wavelength of light used to illuminate the sample and the opening angle of the microscope objective, the achievable resolution is limited to around 200 nm. This so-called classical diffraction limit implies that smaller structural features cannot be resolved or separated from each other. As many food structures are ultimately defined by the molecular interactions of single proteins or single molecules, the classical resolution is insufficient to reveal structural details in the (tens of) nanometer range. Intriguingly, recent advancements in imaging techniques originating mostly in the (biomedical) life sciences have been closing the gap, pushing the resolution towards true molecular resolution. In this perspective, we want to highlight some of these emerging techniques and provide an outlook on potential future applications.

Nanocavitation mechanisms in deformed High Density PolyEthylene (HDPE) using synchrotron radiation NanoTomography

The nanocavitation distribution in deformed High Density PolyEthylene (HDPE) was investigated. A flat notched sample of HDPE, deformed under steady crosshead speed up to the end of the stress softening, was observed by classical synchrotron parallel beam microtomography and magnified holotomography with a true spatial resolution of 0.7 μm and 0.05 μm, respectively. Image processing, 3D image segmentation and 2D-Fast Fourier Transform, was shown to lead to quantification of void volume fraction and void size distributions. Nanocavitation morphology and distribution in HDPE reveal to be similar to cavitation mechanisms in other semi-crystalline polymers, but at a different scale. While a slight decrease of the void diameter was seen, from the end of the neck shoulder to the extended neck region, a significant increment of the height was evidenced. Furthermore, the results provide compelling evidence of morphology transition of cavities within the test sample from an oblate to a prolate geometry.

Phase Resolution of Heterozygous Sites in Diploid Genomes is Important to Phylogenomic Analysis under the Multispecies Coalescent Model.

Genome sequencing projects routinely generate haploid consensus sequences from diploid genomes, which are effectively chimeric sequences with the phase at heterozygous sites resolved at random. The impact of phasing errors on phylogenomic analyses under the multispecies coalescent (MSC) model is largely unknown. Here, we conduct a computer simulation to evaluate the performance of four phase-resolution strategies (the true phase resolution, the diploid analytical integration algorithm which averages over all phase resolutions, computational phase resolution using the program PHASE, and random resolution) on estimation of the species tree and evolutionary parameters in analysis of multilocus genomic data under the MSC model. We found that species tree estimation is robust to phasing errors when species divergences were much older than average coalescent times but may be affected by phasing errors when the species tree is shallow. Estimation of parameters under the MSC model with and without introgression is affected by phasing errors. In particular, random phase resolution causes serious overestimation of population sizes for modern species and biased estimation of cross-species introgression probability. In general, the impact of phasing errors is greater when the mutation rate is higher, the data include more samples per species, and the species tree is shallower with recent divergences. Use of phased sequences inferred by the PHASE program produced small biases in parameter estimates. We analyze two real data sets, one of East Asian brown frogs and another of Rocky Mountains chipmunks, to demonstrate that heterozygote phase-resolution strategies have similar impacts on practical data analyses. We suggest that genome sequencing projects should produce unphased diploid genotype sequences if fully phased data are too challenging to generate, and avoid haploid consensus sequences, which have heterozygous sites phased at random. In case the analytical integration algorithm is computationally unfeasible, computational phasing prior to population genomic analyses is an acceptable alternative. [BPP; introgression; multispecies coalescent; phase; species tree.]

Intensity correlation OCT is a classical mimic of quantum OCT providing up to twofold resolution improvement

Quantum Optical Coherence Tomography (Q-OCT) uses quantum properties of light to provide several advantages over its classical counterpart, OCT: it achieves a twice better axial resolution with the same spectral bandwidth and it is immune to even orders of dispersion. Since these features are very sought-after in OCT imaging, many hardware and software techniques have been created to mimic the quantum behaviour of light and achieve these features using traditional OCT systems. The most recent, purely algorithmic scheme—an improved version of Intensity Correlation Spectral Domain OCT named ICA-SD-OCT—showed even-order dispersion cancellation and reduction of artefacts. The true capabilities of this method were unfortunately severely undermined, both in terms of its relation to Q-OCT and its main performance parameters. In this work, we provide experimental demonstrations as well as numerical and analytical arguments to show that ICA-SD-OCT is a true classical equivalent of Q-OCT, more specifically its Fourier domain version, and therefore it enables a true two-fold axial resolution improvement. We believe that clarification of all the misconceptions about this very promising algorithm will highlight the great value of this method for OCT and consequently lead to its practical applications for resolution- and quality-enhanced OCT imaging.

Practical measurement of the energy resolution for meV-resolved inelastic X-ray scattering.

Several different ways of measuring the energy resolution for meV-resolved inelastic X-ray scattering (IXS) are compared: using scattering from poly(methyl methacrylate), PMMA, using scattering from borosilicate glass (Tempax), and using powder diffraction from aluminium. All of these methods provide a reasonable first approximation to the energy resolution, but, also, in all cases, inelastic contributions appear over some range of energy transfers. Over a range of ±15 meV energy transfer there is good agreement between the measurements of PMMA and Tempax at low temperature, and room-temperature powder diffraction from aluminium, so we consider this to be a good indication of the true resolution of our ∼1.3 meV spectrometer. The resolution over a wider energy range is self-consistently determined using the temperature, momentum and sample dependence of the measured response. The inelastic contributions from the PMMA and Tempax, and their dependence on momentum transfer and temperature, are then quantitatively investigated. The resulting data allow us to determine the resolution of our multi-analyzer array efficiently using a single scan. The importance of this procedure is demonstrated by showing that the results of the analysis of a spectrum from a glass are changed by using the properly deconvolved resolution function. The impact of radiation damage on the scattering from PMMA and Tempax is alsodiscussed.

Single-atom level determination of 3-dimensional surface atomic structure via neural network-assisted atomic electron tomography

Functional properties of nanomaterials strongly depend on their surface atomic structures, but they often become largely different from their bulk structures, exhibiting surface reconstructions and relaxations. However, most of the surface characterization methods are either limited to 2D measurements or not reaching to true 3D atomic-scale resolution, and single-atom level determination of the 3D surface atomic structure for general 3D nanomaterials still remains elusive. Here we demonstrate the measurement of 3D atomic structure at 15 pm precision using a Pt nanoparticle as a model system. Aided by a deep learning-based missing data retrieval combined with atomic electron tomography, the surface atomic structure was reliably measured. We found that <100> and <111> facets contribute differently to the surface strain, resulting in anisotropic strain distribution as well as compressive support boundary effect. The capability of single-atom level surface characterization will not only deepen our understanding of the functional properties of nanomaterials but also open a new door for fine tailoring of their performance.

Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline.

Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-m6A antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single-nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.

Wind direction retrieval from Sentinel-1 SAR images using ResNet

This paper introduces a novel approach to estimate the wind direction over the sea from Synthetic Aperture Radar (SAR) images without any external information. The method employs deep residual network (ResNet), a variant of Convolutional Neural Network, to obtain high resolution (2 km by 2 km) aliased wind direction fields. Forty-seven SAR images of the European Space Agency satellites Sentinel-1 have been processed with ResNet, previously trained with other fifteen images. The areas of interest are the Mediterranean Sea and the Persian Gulf, two regional seas where the SAR images often present complex patterns associated to the wind field spatial structure reporting traces of the interaction with coastal orography, hence valuable test sites to evaluate the performance of the methodology here proposed. Statistical analysis was carried out comparing the SAR-derived wind directions with those from ECMWF atmospheric model, ASCAT scatterometer and in-situ gauges. It reports biases β of -1.1°, 2.4° and -4.6° respectively, and centered root mean square difference cRMSd<21°, consistent with the benchmark obtained comparing scatterometer with ECMWF wind directions over the areas imaged by SAR (β 2.1°, cRMSd=19°). These results are relevant because they include the coastal data not accounted in the benchmark. Analysis of selected cases showed that SAR-derived wind fields reproduce meteorological situations characterized by strong divergence. Notably, our ResNet is able to estimate the wind direction even in the absence of wind streaks on the SAR images and in presence of convective turbulence structures, atmospheric lee waves, and ships. Furthermore, the model is also able to derive the wind field over small areas, as the example of Venice lagoon has shown. Detailed analysis of selected cases raised the issue of the lack of data with true spatial resolution of ≈2 km and within half hour from the satellite pass time necessary for exhaustive comparisons.

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

Simultaneous 2D filtered Rayleigh scattering thermometry and stereoscopic particle image velocimetry measurements in turbulent non-premixed flames

The current work presents simultaneous high-resolution temperature and three-component velocity measurements taken in a series of turbulent (Re = 10,000, 20,000, and 30,000) piloted, non-premixed jet flames using filtered Rayleigh scattering (FRS) thermometry and stereoscopic particle image velocimetry (sPIV). This manuscript details the experimental approach with a key focus on the experimental protocol and specific challenges unique to simultaneous single-shot FRS/sPIV measurements in turbulent non-premixed flames. Specific areas of discussion include the experimental particle scattering rejection for FRS measurements, elimination of signal “crosstalk” between FRS and PIV channels, tracer particle selection, data processing for noise removal, and spatial resolution for both the temperature and velocity measurements. Sample joint temperature/velocity fields are presented highlighting interactions between the flow turbulence and the temperature fields, and a detailed statistical assessment of the temperature and velocity measurements demonstrates the quantitative nature of the results. Dissipation spectra from the measured temperature and velocity fluctuations are used to assess the true spatial resolution of the measurements. Results indicate that the sPIV measurements are resolved well into the dissipative range and the highest spatial frequencies (smallest dissipative scales) are resolved for the temperature measurements under all flame cases and measurement locations. The current joint FRS/sPIV approach enables the first simultaneous single-shot temperature/velocity imaging in turbulent non-premixed flames.

Sample container for high-resolution neutron imaging of spent nuclear fuel cladding sections.

In this work, for the first time, high-resolution neutron imaging (true spatial resolution of 13 μm) is used for irradiated nuclear fuel cladding, applying an adapted procedure for transfer, handling, and measurements of highly radioactive samples in combination with the neutron microscope detector at Paul Scherrer Institut. A sample container referred to as an active box for high-resolution neutron imaging of highly active spent nuclear fuel cladding sections was developed. Sections of unirradiated and irradiated cladding of duplex type, having a liner, with hydrogen average concentrations of 420 wppm and 450 wppm were investigated using this device. The irradiated cladding originated from a fuel rod operated for five cycles in a Swiss pressurized water reactor. The irradiated cladding sample was measured inside the active box. Long circumferential hydride accumulations were revealed together with notable hydride precipitation at the liner-substrate interface. Measurements of the unirradiated cladding in air and inside the active box delivered consistent results, confirming the applicability of the developed device for high-resolution neutron imaging.

NeXT-Grenoble, the Neutron and X-ray tomograph in Grenoble

NeXT-Grenoble is a Neutron and X-ray Tomograph launched from the collaboration between the Universite Grenoble Alpes (UGA) and the Institut Laue Langevin (ILL). The design started in February 2016 shortly followed by its construction at ILL. A first version of the instrument has been opened to users since October 2016.One of the peculiarities of the instrument is that, as suggested by the name, the instrument allows the acquisition of truly simultaneous Neutron and X-ray tomographies, taking advantage of the high complementarity of these two beams.Also by virtue of the uniquely high flux at the ILL, the instrument can provide unprecedented spatial resolution (around 4μm true resolution, in few tens of seconds) and acquisition speed (1.5 s tomographies).This contribution details the adopted technical solutions and performances achieved, reviews some of the key published results and outlines the future direction of the instrument.

Design of High-Performance Photon-Number-Resolving Photodetectors Based on Coherently Interacting Nanoscale Elements

A number of applications in basic science and technology would benefit from high fidelity photon number resolving photodetectors. While some recent experimental progress has been made in this direction, the requirements for true photon number resolution are stringent, and no design currently exists that achieves this goal. Here we employ techniques from fundamental quantum optics to demonstrate that detectors composed of subwavelength elements interacting collectively with the photon field can achieve high-performance photon number resolution. We propose a new design that simultaneously achieves photon number resolution, high efficiency, low jitter, low dark counts, and high count rate. We discuss specific systems that satisfy the design requirements, pointing to the important role of nanoscale device elements.

Automatic segmentation of true color sectioned images using FMRIB Software Library: First trial in brain, gray matter, and white matter.

Three-dimensional (3D) models of the brain made from magnetic resonance images (MRI) are used in various medical fields. 3D models assembled from grayscale color and low-resolution can be complemented with true color sectioned images of the Visible Korean. The purpose of this study is to apply the MRI automatic segmentation technique to the sectioned images. 3D models of the sectioned images, which have true color and high resolution, can be produced without manual segmentation. The Brain Extraction Tool and the Automated Segmentation Tool of the FMRIB Software Library (FSL) were chosen for automatic segmentation. Using those tools, true color sectioned images were reconstructed from gray 3D models of brain, gray matter, and white matter. Color 3D models of those structures were generated from the gray 3D models using MRIcroGL. The color 3D models made from the sectioned images revealed details of brain anatomy that could not be observed on the 3D models from MRI. This trial suggests that convergence of the MRI segmentation technique with color sectioned images is a time-efficient method for producing color 3D models of various structures. In future, the method of this study will be used for various sectioned images of cadavers. The resulting color sectioned images and 3D models will be made available to other researchers.

MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells.

The ultimate goal of biological super-resolution fluorescence microscopy is to provide three-dimensional resolution at the size scale of a fluorescent marker. Here we show that by localizing individual switchable fluorophores with a probing donut-shaped excitation beam, MINFLUX nanoscopy can provide resolutions in the range of 1 to 3 nm for structures in fixed and living cells. This progress has been facilitated by approaching each fluorophore iteratively with the probing-donut minimum, making the resolution essentially uniform and isotropic over scalable fields of view. MINFLUX imaging of nuclear pore complexes of a mammalian cell shows that this true nanometer-scale resolution is obtained in three dimensions and in two color channels. Relying on fewer detected photons than standard camera-based localization, MINFLUX nanoscopy is poised to open a new chapter in the imaging of protein complexes and distributions in fixed and living cells.