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Principal Investigator: James Cherry
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2023
Award: $297,252
Funding agency: National Institute of Allergy and Infectious Diseases
The project particularly aims to use state of the art instrumentation to support the needs for COVID19 research involving flow cytometry.
Increasing number of NIH investigators are transitioning to complex assays for cell analysis. However, sorters currently lack the ability to translate these panels directly into cell sorting for further downstream assays. Under FY23, the flow cytometry section (FCS) within RTB developed new capacities for cell analysis and high-dimensional cell sorting for fast and precise identification of rare cell subsets based on multiple parameters and lasers. This development allowed to open new possibilities for parallel cell analysis and cell isolation based on up to 30 parameter. These combined features have been key for the successful isolation and molecular characterization of very rare (<0.05%) B cells specific for different SARS-Cov2-cells within from unique clinical samples(Dacon et al. 2023). Once optimized, this strategy can easily be shared within a broad portfolio of DIR projects aiming to develop therapeutic strategies for COVID19 and other emerging infectious diseases.
We recently demonstrated that aerosols can be generated during certain operations and proposed BSC enclosure for analysis of infectious samples (Aspland et al 2021). Under FY 23, we completed the setup and a novel cytometry platform contained in a Class II BSC for safe analysis of infectious samples in BSL-3 laboratory. The integration of this platform into a BSL3 lab environment will allow DIR investigators to perform analysis of cellular perturbations in live cells. These findings are expected to provide new insights into cellular mechanisms of host pathogen interactions and to accelerate development of novel therapeutics for COVID19.
Finally, recent advances in spectral technology allows to collect light through the entire spectrum and to objectively manage overlap between fluorescence spectra thereby improving data accuracy compared to conventional flow cytometry. The acquisition of a spectral cell sorter allowed matching and direct transition of complex experimental protocols (based on 40 colors and beyond) currently used by multiple DIR investigators using the Cytek Spectral Analyzer platform. Providing new capabilities for combined ultra-deep immunophenotyping and sorting, will be key to increase understanding of immunes responses to pathogens including SARS-CoV-2, while ensuring continuity in developing this cutting-edge technology.
The Biological Imaging Section (BIS) of RTB, provides expertise, instruction, and instrumentation in optical microscopy to all NIAID investigators and therefore has a history of providing novel technologies when they become available. Staff members are available to assist in experimental design and in the collection and analysis of the high-quality images of living and fixed material. Techniques include confocal, fluorescence, intravital and video microscopy, as well as post collection quantification and deconvolution. Acquisition of state-of-the-art technologies promotes the overall mission of the DIR, NIAID by providing tools to advance projects in basic and clinical research. Imaging has become central to many DIR studies in the field of parasitology, virology, and immunology. In particular, confocal microscopy with its high resolution and multiple channels of data is most appropriate for collecting images of multiply labelled tissue sections or cells. These data are used for determining colocalization of proteins, expression levels of tagged molecules, and location of parasites. At the present time collection of data from samples with many probes (up to 10) over large areas of tissue has become a priority to understanding the immune system.
In order to continue to provide for the needs of NIAID researchers and enable advanced studies of SARS-CoV-2 lung tissue studies, the RTB upgraded and expanded its instruments.
A Leica Stellaris confocal microscope was purchased to replace an existing SP5 confocal microscope that became outdated. The new instrument will be based on a white light laser with expanded excitation range of 440-790nm allowing the use of a much wider range of probes. The new detectors are twice as sensitive, so low light experiments will be possible.
The new state-of-the-art equipment has modern imaging capabilities, including more sophisticated equipment and analysis software, which is required to pursue and finalize advanced research projects including SARS-CoV-2 studies.
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