Nanometer-scale Distribution Research Articles

Nanometer-scale distribution of PD-1 in the melanoma tumor microenvironment.

The nanometer-scale spatial organization of immune receptors plays a role in cell activation and suppression. While the connection between this spatial organization and cell signaling events is emerging from cell culture experiments, how these results translate to more physiologically relevant settings like the tumor microenvironment remains poorly understood due to the challenges of high-resolution imaging in vivo. Here we perform super-resolution immunofluorescence microscopy of human melanoma tissue sections to examine the spatial organization of the immune checkpoint inhibitor programmed cell death 1 (PD-1). We show that PD-1 exhibits a variety of organizations ranging from nanometer-scale clusters to more uniform membrane labeling. Our results demonstrate the capability of super-resolution imaging to examine the spatial organization of immune checkpoint markers in the tumor microenvironment, suggesting a future direction for both clinical and immunology research.

Phosphorus Species in Deep-Sea Carbonate Deposits: Implications for Phosphorus Cycling in Cold Seep Environments

Phosphorus (P) is an important nutrient for biological communities in cold seeps. However, our knowledge on the source, species, and cycling of P in cold seep environments is limited. In this study, the concentration, species, and micro to nanometer scale distribution of P in seep carbonates were examined at three deep-sea cold seeps in the South China Sea and East China Sea. The Ca-P accounts for the largest proportion of P—followed by detrital-P, Fe-P, organic-P, and exchangeable-P. The distribution patterns of Ca-P, detrital-P, and organic-P in the seep carbonates differ from one another, as shown by elemental mapping with NanoSIMS and scanning electron microscopy. The covariation of P with Ca and C reveals that Ca-P co-precipitates with Ca-carbonate, which is linked to the process of sulfate-driven anaerobic oxidation of methane. Organic-P is also observed within biofilm-like organic carbon aggregates, revealing the microbial enrichment of P by fluids in the process of anaerobic oxidation of methane. P with a granulated morphology was identified as detrital-P derived from deep sediments. Most importantly, it is evident that Ca-P is positively correlated to the Fe content in all the seep carbonates. This indicates the likelihood that the dissolved P in cold-seep fluids is released primarily from Fe oxides through Fe-driven anaerobic oxidation of methane in deep sediments. These processes associated with different species of P may have significant implications for P geochemical cycling and anaerobic oxidation of methane impelled by Fe and sulfate reduction in cold seep environments.

Detecting Nanoscale Distribution of Protein Pairs by Proximity-Dependent Super-resolution Microscopy.

Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively.

Nanometer-Scale Distribution of a Lubricant Modifier on Iron Films: A Frequency-Modulation Atomic Force Microscopy Study Combined with a Friction Test.

Liquid lubricants used in mechanical applications are low-vapor-pressure hydrocarbons modified with a small quantity of polar compounds. The polar modifiers adsorbed on the surface of sliding solids dominate the friction properties when the sliding surfaces are in close proximity. However, a few methods are available for the characterization of the adsorbed modifiers of a nanometer-scale thickness. In this study, we applied frequency-modulation atomic force microscopy to evaluate the vertical and lateral density distributions of the adsorbed modifier in a real lubricant, namely, poly-α-olefin (PAO) modified with an orthophosphoric acid oleyl ester. The liquid-induced force on the probing tip was mapped on a plane that was perpendicular to the lubricant–iron interface with a force sensitivity on the order of 10 pN. The PAO in the absence of the ester modifier was directly exposed to the film, which produced a few liquid layers parallel to the film surface with layer-to-layer distances of 0.6–0.7 nm. A monomolecular layer of the modifier was intermittently adsorbed with increasing ester concentration in the bulk lubricant, with complete coverage seen at 20 ppm. The C18H35 chains of the oleyl esters fluctuating in the lubricant produced a repulsive force on the tip, which monotonically decayed with the tip-to-surface distance. The dynamic friction coefficient of sliding steel–lubricant–steel interfaces, which was separately determined using a friction tester, was compared with the force map determined on the iron film immersed in the corresponding lubricant. The complete monomolecular layer of the ester modifier on the static lubricant–iron boundary is a requirement for achieving smooth and stable friction at the sliding interface.

Contributed review: Review of integrated correlative light and electron microscopy.

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemically or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.

Gradient and weather resistant hybrid super‐hydrophobic coating based on fluorinated epoxy resin

ABSTRACTA weather resistant super‐hydrophobic coating that can offer good substrate adhesion and yet to be easily processed at large scale can be of practical use in emerging fields of self‐cleaning and anti‐icing paint, combing all these properties together remains challenging task. Here we describe a composite coating composed of a fluorinated epoxy resin emulsion with embedded in situ surface‐modified dual‐scale nano‐silica, which displayed durable super‐hydrophobicity and excellent adhesive strength. The as‐prepared coating possesses water contact angle of 158.6 ± 1°, sliding angle around 3.8 ± 0.2° which remain stable even under acidic/alkaline, heat/cool, and accelerated aging treatment. The results demonstrate that surface roughness had a micron‐ and nanometer scale distribution with increased particle loading beyond 40 wt %. Through quantitative comparison of surface Attenuated Total Reflection (ATR) with bulk FT‐IR transmission spectra, a gradient coating with surface enrichment of hydrophobic groups was determined. The air‐side fluorinated polysiloxane‐rich layer endows coating with weather‐resistance and ultra‐hydrophobicity while bottom epoxy resin layer enhances substrate adhesion. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40955.

Mineral Distributions at the Developing Tendon Enthesis

Tendon attaches to bone across a functionally graded interface, “the enthesis”. A gradient of mineral content is believed to play an important role for dissipation of stress concentrations at mature fibrocartilaginous interfaces. Surgical repair of injured tendon to bone often fails, suggesting that the enthesis does not regenerate in a healing setting. Understanding the development and the micro/nano-meter structure of this unique interface may provide novel insights for the improvement of repair strategies. This study monitored the development of transitional tissue at the murine supraspinatus tendon enthesis, which begins postnatally and is completed by postnatal day 28. The micrometer-scale distribution of mineral across the developing enthesis was studied by X-ray micro-computed tomography and Raman microprobe spectroscopy. Analyzed regions were identified and further studied by histomorphometry. The nanometer-scale distribution of mineral and collagen fibrils at the developing interface was studied using transmission electron microscopy (TEM). A zone (∼20 µm) exhibiting a gradient in mineral relative to collagen was detected at the leading edge of the hard-soft tissue interface as early as postnatal day 7. Nanocharacterization by TEM suggested that this mineral gradient arose from intrinsic surface roughness on the scale of tens of nanometers at the mineralized front. Microcomputed tomography measurements indicated increases in bone mineral density with time. Raman spectroscopy measurements revealed that the mineral-to-collagen ratio on the mineralized side of the interface was constant throughout postnatal development. An increase in the carbonate concentration of the apatite mineral phase over time suggested possible matrix remodeling during postnatal development. Comparison of Raman-based observations of localized mineral content with histomorphological features indicated that development of the graded mineralized interface is linked to endochondral bone formation near the tendon insertion. These conserved and time-varying aspects of interface composition may have important implications for the growth and mechanical stability of the tendon-to-bone attachment throughout development.

Performance of SPEEK based polymer–nanoclay inorganic membrane for DMFC

Sulfonated poly (ether ether ketone) (SPEEK) membrane with degree of sulfonation (DS) of 63% was modified by adding various amounts of Cloisite15A ® clay. The effect of clay loading on the physical properties of SPEEK nanocomposite membranes was investigated. SPEEK mixed with 2.5 wt.% of Cloisite15A ® (SP63/2.5Cl) has been identified as the best composite formulation in this study. Subsequently, various amounts of 2,4,6-triaminopyrimidine (TAP) were further incorporated into SP63/2.5Cl formulation. The effect of TAP incorporation on the physical and performance properties of the membranes was studied. Nanometer scale distribution of Cloisite15A ® in the SPEEK polymer matrix has been achieved by the assistance of TAP, as observed by the field emission scanning electron microscopy (FESEM). A complete exfoliated nanocomposite structure of SP63/2.5Cl/5.0TAP was confirmed by X-ray diffraction (XRD). The increase of Cloisite15A ® content in the SPEEK membrane has dramatically improved the swelling behavior and barrier properties towards methanol. The SP63/2.5Cl/5.0TAP polymer–clay nanocomposite membrane served as a promising alternative proton exchange membrane (PEM) for DMFC applications with superior DMFC performance compared to commercial Nafion ®112.

Deducing Kinetochore Protein Distributions along and Around a Microtubule Plus-End from FRET Measurements

The eukaryotic kinetochore is a sophisticated motor that moves and segregates chromosomes during cell division. As a microtubule-based motor, the kinetochore is distinct from all other motor proteins and microtubule-associated proteins: it attaches end-on to ∼ 40 nm zone behind the plus-end of one or more microtubules, and it acts as a force coupler rather than an active force generator. These characteristics ensure that chromosome movement is tightly coupled with polymer growth and shortening at the plus-end. They also strongly suggest that kinetochore protein architecture, the distributions of the microtubule-binding kinetochore proteins along and around the microtubule plus-end and their dynamics, dictate the molecular mechanisms that generate movement. We have developed a FRET-based approach to determine the nanometer-scale distributions of multiple copies of the three principle microtubule-binding kinetochore complexes: Dam1, Ndc80, and Spc105. Sensitized emission measurements are carried out in metaphase and anaphase budding yeast (Saccharomyces cerevisiae) cells expressing kinetochore proteins labeled with Cerulean (donor) and Venus (acceptor) at desired locations. Preliminary measurements probing the distribution of the Ndc80 complex molecules show that the microtubule-binding head domains of adjacent Ndc80 molecules are ∼ five nm apart, but this distance increases significantly at the other end of the molecule. The Dam1 complex is located more than 10 nm away from these head domains of Ndc80. We are developing Monte Carlo approaches to simulate the energy transfer processes among a known number of donor and acceptor molecules using known protein structures of some of the kinetochore proteins and the cylindrical microtubule lattice. These simulations will identify molecular distributions consistent with the FRET data, and illuminate the structural basis for microtubule end-coupled movement of the kinetochore.

Nanometer-scale distribution of field emission current from the arc-prepared carbon thin film

In order to clarify the mechanism of low-macroscopic-field (LMF) electron emission from carbon related materials, atomistic properties including local tunneling barrier height (LBH) and field emission (FE) current distributions of the arc-prepared carbon thin film have been measured by using scanning tunneling microscopy. From the LBH images, it is found that the carbon thin film surface consists of nanometer-scale graphite grains whose crystallinities and electronic properties are different although the local work function of the film is rather homogeneous. The obtained FE images show that the FE currents are varied more than one order of magnitude, depending on the grains, and that the FE current is higher at the edge of the individual grains. The authors cannot find any specific emission sites that give extremely higher emission currents, indicating that the specific emission sites such as high-aspect-ratio nanoprotrusions or atomistic defects are not responsible to the LMF electron emission from carbon related materials.

Native surface structure of natural soil particles determined by combining atomic force microscopy and X-ray photoelectron spectroscopy

A procedure was developed for handling natural soil particles and probing their native surface structure by atomic force microscopy (AFM) under water. This procedure was used to investigate the nanometer scale organisation of organic matter at the surface of sand particles taken from three soil horizons. The latter were selected for the contrasted properties of their organic matter, namely Podzol E and Bh horizons and a Cambisol A–B horizon. The presence of an adsorbed layer was visualised at the surface of Podzol Bh and Cambisol particles in the form of aggregated structures that interacted with the AFM probe. Surface analysis by X-ray photoelectron spectrometry (XPS) confirmed the carbonaceous nature of this adsorbed layer. Displacement of organic matter by the scanning probe was directly evidenced for Podzol Bh sand particles. Such displacement was not observed for Cambisol particles. A dramatic effect of drying on the concentration, nanometer scale distribution and properties of the adsorbed organic matter was clearly demonstrated by combining AFM imaging and XPS analysis. The procedure developed here gives access to direct, nanoscale information of the surface structure of sand particles and offers promising prospects for the characterisation of other environmentally-relevant particles in native conditions.

Core pinning by intragranular nanoprecipitates in polycrystallineMgCNi3

The nanostructure and magnetic properties of polycrystalline MgCNi_3 were studied by x-ray diffraction, electron microscopy, and vibrating sample magnetometry. While the bulk flux-pinning force curve F_p(H) indicates the expected grain-boundary pinning mechanism just below T_c = 7.2 K, a systematic change to pinning by a nanometer-scale distribution of core pinning sites is indicated by a shift of F_p(H) with decreasing temperature. The lack of scaling of F_p(H) suggests the presence of 10 to 20% of nonsuperconducting regions inside the grains, which are smaller than the diameter of fluxon cores 2xi at high temperature and become effective with decreasing temperature when xi(T) approaches the nanostructural scale. Transmission electron microscopy revealed cubic and graphite nanoprecipitates with 2 to 5 nm size, consistent with the above hypothesis since xi(0) = 6 nm. High critical current densities, more than 10^6 A/cm^2 at 1 T and 4.2 K, were obtained for grain colonies separated by carbon. Dirty-limit behavior seen in previous studies may be tied to electron scattering by the precipitates, indicating the possibility that strong core pinning might be combined with a technologically useful upper critical field if versions of MgCNi_3 with higher T_c can be found.