Infectious Diseases Collaborative Technological Research Resources- RML

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

In 2022, 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. Electron Microscopy The RML Microscopy (EM) Unit provides expertise in advanced electron microscopy (EM)-related techniques and technologies to support the ultra-structural imaging needs of the DIR scientists both in Bethesda and at the Rocky Mountain Laboratories (RML). The facility provides sample preparation, imaging, and analysis, ranging from basic structural studies and immune localization of selected antigens to high resolution and three-dimensional analyses for a wide array of specimens. Addition of focused ion beam scanning electron microscopy (FIB-SEM), light microscopy (LM), digital spatial profiling system and expansion of the cryo-EM technologies both at the Bethesda and RML campuses provide greater access for DIR scientists to advanced imaging technologies and spatial genomic studies. The facility provides sample preparation, imaging, and analysis, ranging from basic structural studies and immune-localization of selected antigens to high resolution and three-dimensional analyses for a wide array of specimens. Optimal preservation of specimens is one of the major challenges in the field of EM, and the EM Unit uses a variety of methods, protocols, and equipment to accommodate different preparative and imaging needs. Conventional techniques for sample preparation inherently introduce structural changes in specimens, which can lead to artifacts. The EM Unit has put much effort into this critical first step as evidenced by the development of application-specific chemical and cryogenic preparative techniques to optimally preserve structures of interest for visualization and interpretation. These techniques in combination with new advanced imaging technologies and the ability to plunge freeze for near native state preservation, now allows visualization of macromolecular complexes, viruses, bacteria, and host-pathogen interactions at high resolution and in three dimensions. Concurrently, our bioinformatics component continues to evolve for high-resolution analyses of increasingly complex structures, and more efficient and comprehensive data collection for higher and better throughput. The broad educational background of the EM Unit staff, along with the opportunity to participate in microscopy meetings and hands-on workshops have been critical for expanding the repertoire of techniques. The Unit has expertise for both tomographic data collection and high resolution analysis tools, protocol development especially for immunology, optimizing parameters for techniques including data collection schemes, microwave-assisted freeze substitution (MWFS), and correlative light and electron microscopy (CLEM). Collaborations with various experts in structural biology and cryo-electron tomography (Cryo-ET) continue to provide both analytical assistance vital for determining viral and macromolecular structures, and automated tomographic alignments for improving database efficiency and workflow. In particular, the light microscopy operations now have expanded with extensive experience in studying bacteria, viruses, and human parasites and their interactions with the host, using an array of light microscopy technologies including live, low-light imaging, wide-field/deconvolution, confocal, light-sheet, and structured illumination super-resolution microscopy. New personnel include interns and contract hires who bring both microscopy and data management expertise. On-going developments in both techniques and technology include but not limited to; 1) optimizing specimen preparation for immunology, 2) light microscopy (LM), 3) plunge freezing, 4) application of machine-learning, 5) light microscopy upgrades, 6) high resolution cameras and whole cell visualization though focused-ion beam scanning electron microscopy (FIB-SEM). All have allowed for an overall expansion of EM capabilities in several areas including significant improvement in 3-D visualization and resolution potential at 3 Angstroms. Simultaneously, our imaging-informatics component continually evolves with high -resolution analyses of increasingly complex structures, the ability to quantify various types of data, along with more efficient and comprehensive data collection for higher resolution and throughput. Flow Cytometry The Flow Cytometry Section (FCS) provides the DIR with application-specific flow cytometric technologies, including instrumentation for high speed cell sorting and multi-color analysis. In addition, the FCS provides training, consultation, method development, and analysis for experiments involving flow cytometry. This mission is accomplished through the efforts of dedicated staff with >200 years of cumulated flow cytometry experience. FCS also provides cost- and time-efficient methods of obtaining fluorescent-labeled proteins or antibodies. Resources available include custom hybridoma expansion; antibody purification from ascites, antisera, and hybridoma supernatant; and coupling of purified antibodies and proteins to various fluorochromes. Major goals are to provide access to state-of-the art technologies, to help design and run experiments, to facilitate data interpretation and to provide results that are of consistent high quality. Project-specific research applications are developed for flow cytometry analysis and sorting in BSL-2, BSL-3 and BSL-4 environments. RML Genomics Research The Genomic Research Section (GRS) provides state-of-the-art next-generation sequencing, and other massively parallel genomic technologies to intramural investigators. In addition to managing high-end instrumentation, computers, and protocols involved in genome-wide assays, the GTS offers statistical analysis and bioinformatics for data interpretation. This support involves close collaboration with the investigators in both experimental design and data analysis to best utilize the power of these applications, as well as to convey a better understanding of the technology. GTS develops project-specific research applications for many analysis platforms at the DNA, RNA and protein level. Statistical analysis, data management, and data mining solutions are available as well, including from experimental concept to publication and public data submission. The Section maintains instrumentation and resources for sequencing applications using the Illumina high throughput sequencing (NGS), including RNA-seq, DNA-seq, ChIP-seq, ATAC-seq, Rep-seq, and single-cell RNA profiling and many others. Some microarray applications such as single-cell Western and protein arrays are supported as well. Visual and Medical Arts The Visual and Medical Arts Unit (VMA) at Rocky Mountain Laboratories (RML) provides highly specialized and advanced technologies to support the Division of Intramural Research (DIR) program. The unit offers a wide variety of digital technologies, involving a comprehensive suite of illustration, design services, photography, 3D animation, video, and new media to document scientific findings. In addition, the VMA provided scientific visualizations to the Office of the Director, NIAID for presentation to scientists, the public as well as US lawmakers. This unit works in close collaboration with the research community.