Project description:Retrieving high-content gene-expression information while retaining three-dimensional (3D) positional anatomy at cellular resolution has been difficult, limiting integrative understanding of structure and function in complex biological tissues. We developed and applied a technology for 3D intact-tissue RNA sequencing, termed STARmap (spatially-resolved transcript amplicon readout mapping), which integrates hydrogel-tissue chemistry, targeted signal amplification, and in situ sequencing. The capabilities of STARmap were tested by mapping 160 to 1020 genes simultaneously in sections of mouse brain at single-cell resolution with high efficiency, accuracy, and reproducibility. Moving to thick tissue blocks, we observed a molecularly defined gradient distribution of excitatory-neuron subtypes across cubic millimeter-scale volumes (>30,000 cells) and a short-range 3D self-clustering in many inhibitory-neuron subtypes that could be identified and described with 3D STARmap.
Project description:The powerful and specific molecular-recognition system present in the base-pairing of DNA allows for the design of a plethora of nanostructures. In this work, the crystallization of a self-assembling three-dimensional B-DNA nanostructure is described. The DNA nanostructure consists of six single-stranded oligonucleotides that hybridize to form a three-dimensional tetrahedron of 80 kDa in molecular mass and 20 bp on each edge. Crystals of the tetrahedron have been successfully produced and characterized. These crystals may form the basis for an X-ray structure of the tetrahedron in the future. Nucleotide crystallography poses many challenges, leading to the fact that only 1352 X-ray structures of nucleic acids have been solved compared with more than 80,000 protein structures. In this work, the crystallization optimization for three-dimensional tetrahedra is also described, with the eventual goal of producing nanocrystals to overcome the radiation-damage obstacle by the use of free-electron laser technology in the future.
Project description:Organismal colour can be created by selective absorption of light by pigments or light scattering by photonic nanostructures. Photonic nanostructures may vary in refractive index over one, two or three dimensions and may be periodic over large spatial scales or amorphous with short-range order. Theoretical optical analysis of three-dimensional amorphous nanostructures has been challenging because these structures are difficult to describe accurately from conventional two-dimensional electron microscopy alone. Intermediate voltage electron microscopy (IVEM) with tomographic reconstruction adds three-dimensional data by using a high-power electron beam to penetrate and image sections of material sufficiently thick to contain a significant portion of the structure. Here, we use IVEM tomography to characterize a non-iridescent, three-dimensional biophotonic nanostructure: the spongy medullary layer from eastern bluebird Sialia sialis feather barbs. Tomography and three-dimensional Fourier analysis reveal that it is an amorphous, interconnected bicontinuous matrix that is appropriately ordered at local spatial scales in all three dimensions to coherently scatter light. The predicted reflectance spectra from the three-dimensional Fourier analysis are more precise than those predicted by previous two-dimensional Fourier analysis of transmission electron microscopy sections. These results highlight the usefulness, and obstacles, of tomography in the description and analysis of three-dimensional photonic structures.
Project description:A sensitive electrochemical immunosensor was developed for detection of alpha-fetoprotein (AFP) based on a three-dimensional nanostructure gold electrode using a facile, rapid, "green" square-wave oxidation-reduction cycle technique. The resulting three-dimensional gold nanocomposites were characterized by scanning electron microscopy and cyclic voltammetry. A "sandwich-type" detection strategy using an electrochemical immunosensor was employed. Under optimal conditions, a good linear relationship between the current response signal and the AFP concentrations was observed in the range of 10-50 ng/mL with a detection limit of 3 pg/mL. This new immunosensor showed a fast amperometric response and high sensitivity and selectivity. It was successfully used to determine AFP in a human serum sample with a relative standard deviation of <5% (n=5). The proposed immunosensor represents a significant step toward practical application in clinical diagnosis and monitoring of prognosis.
Project description:The polymer electrolyte fuel cell (PEFC) is one of the strongest candidates for a next-generation power source for vehicles which do not emit CO2 gas as exhaust gas. The key factor in PEFCs is the nano-scaled electrochemical reactions that take place on the catalyst material and an ionomer supported by a carbon support. However, because the nano-scaled morphological features of the key materials in the catalyst compound cannot be observed clearly by transmission electron microscopy, improvement of PEFC performance had been approached by an imaginal schematic diagram based on an electrochemical analysis. In this study, we revealed the nano-scaled morphological features of the PEFC electrode in three dimensions and performed a quantitative analysis of the nanostructure by the newly developed 'Combined ADF-STEM tomography technique'. This method combines information from plural annular darkfield detectors with different electron collection angles and can emphasize the difference of the electron scattering intensity between the ionomer and carbon in the cross-sectional image of the reconstructed three-dimensional (3D) data. Therefore, this segmentation method utilizing image contrast does not require a high electron beam current like that used in energy dispersive X-ray analysis, and thus is suitable for electron beam damage-sensitive materials. By eliminating the process of manually determining the thresholds for obtaining classified component data from grayscale data, the obtained 3D structures have sufficient accuracy to allow quantitative analysis and specify the nano-scaled structural parameters directly related to power generation characteristics.
Project description:Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20-60 Pa H(2)O) conditions prevent charging of the uncoated block face. The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50-70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits.
Project description:Mineral inclusions in biomass are attracting increased scrutiny due to their potential impact on processing methods designed to provide renewable feedstocks for the production of chemicals and fuels. These inclusions are often sculpted by the plant into shapes required to support functional roles that include the storage of specific elements, strengthening of the plant structure, and providing a defense against pathogens and herbivores. In situ characterization of these inclusions faces substantial challenges since they are embedded in an opaque, complex polymeric matrix. Here we describe the use of Bragg coherent diffraction imaging (BCDI) to study mineral inclusions within intact maize stalks. Three-dimensional BCDI data sets were collected and used to reconstruct images of mineral inclusions at 50-100 nm resolution. Asymmetries in the intensity distributions around the Bragg peaks provided detailed information about the deformation fields within these crystal particles revealing lattice defects that result in distinct internal crystal domains.
Project description:We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.
Project description:MotivationThe arbor morphologies of brain microglia are important indicators of cell activation. This article fills the need for accurate, robust, adaptive and scalable methods for reconstructing 3-D microglial arbors and quantitatively mapping microglia activation states over extended brain tissue regions.ResultsThick rat brain sections (100-300 µm) were multiplex immunolabeled for IBA1 and Hoechst, and imaged by step-and-image confocal microscopy with automated 3-D image mosaicing, producing seamless images of extended brain regions (e.g. 5903 × 9874 × 229 voxels). An over-complete dictionary-based model was learned for the image-specific local structure of microglial processes. The microglial arbors were reconstructed seamlessly using an automated and scalable algorithm that exploits microglia-specific constraints. This method detected 80.1 and 92.8% more centered arbor points, and 53.5 and 55.5% fewer spurious points than existing vesselness and LoG-based methods, respectively, and the traces were 13.1 and 15.5% more accurate based on the DIADEM metric. The arbor morphologies were quantified using Scorcioni's L-measure. Coifman's harmonic co-clustering revealed four morphologically distinct classes that concord with known microglia activation patterns. This enabled us to map spatial distributions of microglial activation and cell abundances.Availability and implementationExperimental protocols, sample datasets, scalable open-source multi-threaded software implementation (C++, MATLAB) in the electronic supplement, and website (www.farsight-toolkit.org). http://www.farsight-toolkit.org/wiki/Population-scale_Three-dimensional_Reconstruction_and_Quanti-tative_Profiling_of_Microglia_ArborsContactbroysam@central.uh.eduSupplementary informationSupplementary data are available at Bioinformatics online.