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Results for “Cell structure identification”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

SINGLE: Atomic-resolution structure identification of nanocrystals by graphene liquid cell EM

Analysis of the three-dimensional (3D) structures of nanocrystals with solution-phase transmission electron microscopy is beginning to reveal their unique physiochemical properties. We developed a “one-particle Brownian 3D reconstruction method” based on imaging of ensembles of colloidal nanocrystals using graphene liquid cell electron microscopy. Projection images of differently rotated nanocrystals are acquired using a direct electron detector with high temporal (<2.5 ms) resolution and analyzed to obtain an ensemble of 3D reconstructions. Here, we introduce computational methods required for successful atomic-resolution 3D reconstruction: (i) tracking of the individual particles throughout the time series, (ii) subtraction of the interfering background of the graphene liquid cell, (iii) identification and rejection of low-quality images, and (iv) tailored strategies for 2D/3D alignment and averaging that differ from those used in biological cryo–electron microscopy. Our developments are made available through the open-source software package SINGLE.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy

Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength, etc., which influences the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77 K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. Finally, by expressing an inducible roGFP2-PopZ fusion we visualize individual microdomains in the context of their redox environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Optical Microscopy-Guided Laser Ablation Electrospray Ionization Ion Mobility Mass Spectrometry: Ambient Single Cell Metabolomics with Increased Confidence in Molecular Identification

Single cell analysis is a field of increasing interest as new tools are continually being developed to understand intercellular differences within large cell populations. Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is an emerging technique for single cell metabolomics. Over the years, it has been validated that this ionization technique is advantageous for probing the molecular content of individual cells in situ. Here, we report the integration of a microscope into the optical train of the LAESI source to allow for visually informed ambient in situ single cell analysis. Additionally, we have coupled this ‘LAESI microscope’ to a drift-tube ion mobility mass spectrometer to enable separation of isobaric species and allow for the determination of ion collision cross sections in conjunction with accurate mass measurements. This combined information helps provide higher confidence for structural assignment of molecules ablated from single cells. Here, we show that this system enables the analysis of the metabolite content of Allium cepa epidermal cells with high confidence structural identification together with their spatial locations within a tissue.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Time-resolved atomic-resolution Brownian tomography of single nanocrystals reveals size-dependent dynamics

Atomic-resolution structure identification of nanocrystals by graphene liquid cell electron microscopy (GLC-EM) has revealed that small, solubilized platinum nanocrystals consist of an ordered crystalline core surrounded by mobile surface atoms, which dissociate during oxidative etching, resulting in distinct temporal structural states. Requirements imposed by the 3D reconstruction algorithm limit the number of structural states that can be resolved. We introduce a regularized 3D reconstruction algorithm that exploits the redundancy inherent in the experimental data, allowing us to improve the time resolution. Our developments provide a comprehensive molecular picture at unprecedented spatial and temporal resolution of the nonlinear, linear, and fluctuating dynamic phenomena that single nanocrystals undergo during the GLC-EM experiment. We determined atomic structures of 66 temporal structural states, extracted from 15 time trajectories of individual nanocrystals. Large (478 to 698 atoms) and small (<300 atoms) nanocrystals show etching that preserves a stable core, whereas mid-sized (351 to 571 atoms) nanocrystals present dynamics that change the coordination of the core.

Meana-Pañeda, Rubén↗
DOE OSTI2013

Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: Identification of an RGFRRR motif governing fungal cell entry

A highly conserved plant defensin MtDef4 potently inhibits the growth of a filamentous fungus Fusarium graminearum. MtDef4 is internalized by cells of F. graminearum. To determine its mechanism of fungal cell entry and antifungal action, NMR solution structure of MtDef4 has been determined. The analysis of its structure has revealed a positively charged patch on the surface of the protein consisting of arginine residues in its γ-core signature, a major determinant of the antifungal activity of MtDef4. Here, we report functional analysis of the RGFRRR motif of the γ-core signature of MtDef4. The replacement of RGFRRR to AAAARR or to RGFRAA not only abolishes fungal cell entry but also results in loss of the antifungal activity of MtDef4. MtDef4 binds strongly to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Mutations of RGFRRR which abolish fungal cell entry of MtDef4 also impair its binding to PA. Our results suggest that RGFRRR motif is a translocation signal for entry of MtDef4 into fungal cells and that this positively charged motif likely mediates interaction of this defensin with PA as part of its antifungal action.

antifungal agent↗
DOE OSTI2025

Evolution and Degradation Patterns of Electrochemical Cells Based on the Analysis of Interfacial Phenomena at Li Metal Anode/Electrolyte Interfaces

In this work, we report the results of a theoretical–computational analysis of the solid electrolyte interphase (SEI) growth and degradation dynamics occurring in lithium metal batteries during cycling. We use ab initio-kinetic Monte Carlo simulations to generate a synthetic data set, which is analyzed by machine learning methods. We aim to determine: (i) how modifications in interfacial interaction energies between solid electrolyte interphase (SEI) blocks and between Li ions and SEI facets impact the Coulombic efficiency (CE) of the battery and (ii) what factors, including reactions, microscopic transport, and other interfacial events, may lead to cell performance “failure” during prolonged charge and discharge cycles, signaled as a sharp decay in the CE over cycling. The demonstration of our approach is done on a cell including a Li metal surface interfacing with a previously introduced state-of-the-art electrolyte, and the idea can be applied to any electrochemical system. Outcomes include the identification of the leading chemical, physical, and structural variables causing cell failure and relating them to the electrolyte formulation, thus paving the way to future more refined analysis and electrolyte design.

batteries↗
DOE OSTI2014

Visualization of Hierarchical Nanodomains in Polymer/Fullerene Bulk Heterojunction Solar Cells

Here, traditional electron microscopy techniques such as bright-field imaging provide poor contrast for organic films and identification of structures in amorphous material can be problematic, particularly in high-performance organic solar cells. By combining energy-filtered corrected transmission electron microscopy, together with electron energy loss and X-ray energy-dispersive hyperspectral imaging, we have imaged PTB7/ PC 61 BM blended polymer optical photovoltaic films, and were able to identify domains ranging in size from several hundred nanometers to several nanometers in extent. This work verifies that microstructural domains exist in bulk heterojunctions in PTB7/PC 61 BM polymeric solar cells at multiple length scales and expands our understanding of optimal device performance providing insight for the design of even higher performance cells.

14 SOLAR ENERGY↗
DOE OSTI2021

Cryo-electron tomography provides topological insights into mutant huntingtin exon 1 and polyQ aggregates

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Nano-structural analysis of engrafted human induced pluripotent stem cell-derived cardiomyocytes in mouse hearts using a genetic-probe APEX2

Many studies have shown the feasibility of in vivo cardiac transplantation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in animal experiments. However, nano-structural confirmation of the successful incorporation of the engrafted iPSC-CMs including electron microscopy (EM) has not been accomplished, partly because identification of graft cells in EM has proven to be difficult. Using APEX2, an engineered ascorbate peroxidase imaging tag, we successfully localized and analyzed the fine structure of sarcomeres and the excitation contraction machinery of iPSC-CMs 6 months after their engraftment in infarcted mouse hearts. APEX2 made iPSC-CMs visible in multiple imaging modalities including light microscopy, X-ray microscopic tomography, transmission EM, and scanning EM. EM tomography allowed assessment of the differentiation state of APEX2-positive iPSC-CMs and analysis of the fine structure of the sarcomeres including T-tubules and dyads.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Resolving atomistic structure and oxygen evolution activity in nickel antimonates

The oxygen evolution reaction (OER) requires electrodes that are not only catalytically active, but also stable under harsh electrochemical environments to enable efficient, durable technologies. Our recent report of a stable amorphous Ni 0.5 Sb 0.5 O z OER photoanode established Ni–Sb–O as an important system for computational understanding of both the structural and catalytic behavior of these complex oxides. In the present work we show that Ni x Sb 1-x O z with x > 0.33 crystallizes into a previously unknown phase. Guided by experimental X-ray diffraction, here, we use density functional theory calculations to perform a prototype phase search to identify a broad family of stable and metastable mixed rutile and hexagonal-like phases for x = 0.33, 0.50, and 0.66 compositions. For the identified phases, we predict favorable oxygen vacancy formation energies for Ni-rich compositions under the reducing synthesis conditions which match measured Ni K-edge X-ray absorption spectra. The calculated overpotential for the most active site decreases with increasing Ni content, from 0.91 V (x = 0.33) to 0.49 V (x = 0.66), which captures the experimentally observed trend. We find the active site changes from the Ni–O–Sb bridge to a Ni–O–Ni bridge at increasing Ni concentrations, rather than the commonly studied singly under-coordinated sites. Finally, detailed Pourbaix analysis of the identified phases show excellent electrochemical stability, consistent with experimentally measured low metal ion concentrations in the electrolyte of photoelectrochemical cells. Collectively, our consideration of an ensemble of structures enables identification of the most catalytically prolific structural motifs, aiding the understanding of crystalline and amorphous catalysts and elucidating the co-optimization of activity and durability in nickel antimonates.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

The effects of chemical fixation on the cellular nanostructure

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods. - Highlights: • PWS was employed to monitor fixation process for the same cells in vitro. • Dramatic changes in cellular nanostructure were observed after fixation. • However, the population difference of chromatin structure remains after fixation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Theoretical exploration of various lithium peroxide crystal structures in a Li-air battery

We describe a series of metastable Li₂O₂ crystal structures involving different orientations and displacements of the O₂²⁻ peroxy ions based on the known Li₂O₂ crystal structure. Within the vicinity of the chemical potential ΔG ~ 0.20 eV/Li from the thermodynamic ground state of the Li₂O₂ crystal structure (i.e., Föppl structure), all of these newly found metastable Li₂O₂ crystal structures are found to be insulating and high-k materials, and they have a common unique signature of an O₂²⁻ O-O vibration mode (ω ~ 799–865 cm⁻¹), which is in the range of that commonly observed in Li-air battery experiments, regardless of the random O₂²⁻ orientations and the symmetry in the crystal lattice. From XRD patterns analysis, the commercially available Li₂O₂ powder is confirmed to be the thermodynamic ground state Föppl-like structure. However, for Li₂O₂ compounds that are grown electrochemically under the environment of Li-O₂ cells, we found that the XRD patterns alone are not sufficient for structural identification of these metastable Li₂O₂ crystalline phases due to the poor crystallinity of the sample. In addition, the commonly known Raman signal of O₂²⁻ vibration mode is also found to be insufficient to validate the possible existence of these newly predicted Li₂O₂ crystal structures, as all of them similarly share the similar O₂²⁻ vibration mode. However considering that the discharge voltage in most Li-O₂ cells are typically several tenths of an eV below the thermodynamic equilibrium for the formation of ground state Föppl structure, the formation of these metastable Li₂O₂ crystal structures appears to be thermodynamically feasible.

25 ENERGY STORAGE↗