We request funds for the purchase of a shared Zeiss LSM880 confocal microscope with Airyscan detector, which will facilitate multiple NIH-funded projects by providing high-speed super-resolution imaging in three- dimensions throughout living cells and tissues. The instrument will be used to visualize and quantify dynamic macromolecular interactions associated with vesicular/membrane trafficking and exocytosis. These data are critical to ongoing investigations into endosomal sorting and synaptic trafficking, extracellular matrix interactions, cell invasion, and cilia formation amongst others. The Airyscan detector, which facilitates imaging beyond the diffraction limit at a resolution close to Structured Illumination Microscopy (SIM) in all three spatial dimensions (which is critical for these studies) is not currently available at Washington University or the surrounding geographical area. In addition to the Airyscan detector, the supported imaging experiments in living cells require multiple laser excitation and multi-spectral detection, dynamic autofocus, and an incubation system. Nine Major User groups from eight departments of Washington University, spanning the School of Medicine, the College of Arts and Sciences and the School of Engineering and Applied Sciences use imaging techniques to study a broad range of problems in inflammation, neurodegenerative and neurological disorders, ciliopathies, vascular malformation, hearing loss and circadian physiology. All have considerable expertise with imaging-based approaches to quantify vesicular and non-vesicular processes and have critical experimental needs for high-speed super-resolution imaging. This instrument has been configured to meet the specific needs of the major user group, but is expected to impact many other projects as well. To maximize this impact, the instrument will be located in the Washington University Center for Cellular Imaging (WUCCI), and maintained by the WUCCI for the benefit of all Washington University investigators. This space is secure and easily accessible to all major users. The expertise and institutional support for this instrument are exceptional. Dr. James Fitzpatrick, the Scientific Director of WUCCI, and Dr. David Piston, the Chair of Cell Biology & Physiology and head of the WUCCI Advisory Board, are both world-renowned experts in cellular microscopy methods. The WUCCI is a recently created multi-disciplinary collaborative shared technology resource center whose leadership brings over 30 years of combined experience in providing cost-efficient training and support for high quality quantitative cellular imaging to a wide range of NIH-funded users. To create the WUCCI, WUSM has provided an initial investment of over $6M and dedicated over 6,000 square feet of specifically designed lab space. In support of this S10 grant application, WUSM will also commit $50,000 per year for four years to ensure the long-term success of this instrument (see supporting letters).

Public Health Relevance

The exact three-dimensional arrangement of sub-cellular constituents is tremendously important, as are the time-dependent changes in their distribution particularly in response to external signals and in disease. Understanding the spatio-temporal organization of these components is critically important to unraveling the complex dynamic macromolecular interactions that govern vesicular/membrane trafficking, endosomal sorting and exocytosis. Recent advances in microscopy, such as high-efficiency spectral detectors which allow the separation of multiple fluorescent signals, combined with super-resolution microscopy as requested here, allow us to observe such rearrangements in living cells in real-time.

National Institute of Health (NIH)
Office of The Director, National Institutes of Health (OD)
Biomedical Research Support Shared Instrumentation Grants (S10)
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Special Emphasis Panel (ZRG1-CB-T (30)I)
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Horska, Alena
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Washington University
Schools of Medicine
Saint Louis
United States
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