In 2015, this project provides state-of-the-art research technologies that are developed, validated, and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the intramural program. The Genomics Unit provides applications in NGS sequencing, microarray, human and pathogen genotyping, and bioinformatic data analysis. More than 400,000 samples were processed by the Unit in the last 4 years. The Units Capillary DNA sequencing uses the AB 3730XL with 96-well high throughput processing with reads lengths out to 900bps. NGS employs the Illumina HiSeq 2500, and the MiSeq towards small RNA discovery, ChIP-Seq, transcriptomics, exome sequencing, de novo and ref-map genome sequencing, and copy number variation studies. Applications are developed in close collaboration with DIR investigators. The Unit develops project-specific research applications on the Affymetrix microarray platform including custom chip design, experimental design, sample processing and chip processing. Statistical analysis, data management, and data mining solutions are available as well, including from experimental concept to publication and public data submission. qPCR is performed for microarray and NGS data validation, expression analysis, sample optimization, and data support. Several technologies are available for human and pathogen genotyping, ranging from capillary-based re-sequencing, to high throughput targeted SNP genotyping via allelic discrimination assays. NGS for de novo SNP, In/Del, copy number, alternative splice variant analysis and newly expressed region discovery for both human and pathogen genomes is available. The Unit provides bioinformatics support for all of the offered technologies and applications. Flow Cytometry Project-specific research applications are developed for flow cytometry analysis and sorting in BSL-2 and BSL-3 environments. Electron Microscopy The Electron Microscopy Unit develops project-specific research applications in areas of sample preparation and analysis, ranging from basic structural studies to immune-localization of selected antigens for a wide array of specimens. A variety of methods, protocols, and equipment are employed to accommodate different preparative and imaging needs. Recent technological advancements have focused on 1) the introduction and optimization of sophisticated preparative technologies and techniques for improved retention and visualization of labile structures often lost during routine processing and improving structural preservation and 2) the introduction of advanced imaging technologies including high resolution transmission and scanning electron microscopes. The Unit has been developing correlative techniques for examining transient or dynamic events by light microscopy to identify regions of interest, which can then be prepared for visualization by electron microscopy to definitively correlate structures with functional assays. Conventional specimen processing for examination by electron microscopy requires use of chemicals that often extract or alter structures of interest. Cryo-preservation through high pressure freezing followed by chemical exchange at low temperature in a process called freeze substitution, has become the preferred technique allowing retention of fragile structures. It is a lengthy process of replacing vitreous ice in rapidly-frozen hydrated samples with an organic solvent containing fixatives and electron-dense contrasting agents. The EM Unit developed and assessed methods for maintaining cryo conditions in a laboratory microwave processor. Further development resulted in the fabrication of a thermally controllable unit decreasing processing periods from several days to a few hours while achieving excellent structural and antigenic preservation. The Microwave Assisted Freeze Substitution concept resulted in a patent application. The 300 kV TEM microscope for high-resolution 3D biological imaging is configured to have optimal flexibility to respond to the needs of investigators and provide them with the highest quality images. The main goal is to achieve the highest-level resolution possible for the characterization of macromolecular complexes, cellular organelles, viruses, bacteria and other parasites, as well as the ability to observe in three dimensions the host pathogen interactions occurring within eukaryotic cells. These technologies improve investigators ability to relate structure to function, providing information that may identify vaccine targets or other intervention strategies. Current projects include high-resolution imaging and reconstruction of bacteria, viruses, macromolecular complexes and eukaryotic cells. The ability to assign cellular function to structures provides valuable information in the study of host pathogen interactions. Fluorescent labeling antigens of interest or use of green and red fluorescent proteins as genetic markers allows visualization of transient and dynamic events by light microscopy (LM). Although advances in technology have improved resolution by LM, electron microscopy (EM) still provides superior ability for resolving small structures. However, EM provides only a snapshot since specimens are immobilized during specimen processing, limiting information about cellular dynamics. Correlative light and electron microscopy bridges this gap by coupling dynamic or transient information available through LM with the ability to resolve ultrastructural details by EM. Innovative methods have been established by the Unit that enable identification and imaging cells of interest first by confocal laser or epifluorescent microscopy, and then by scanning or transmission electron microscopy. Visual and Medical Arts The Visual and Medical Arts Unit (VMA) at RML provides highly specialized and technical services to the DIRs scientific and support staff. The Unit assists investigators in the use of images, interactive technologies, and animation/simulation to effectively communicate complex science and health topics to a range of audiences in scientific publications and new media platforms. The VMA is integral in providing support for complex web-based applications developed by the RTB. They work closely with scientists on individual projects, as well as produce video and other visualizations for public consumption.
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