This proposal represents a request from a group of NIH-funded investigators for funds to acquire a Zeiss Lightsheet Z.1 Fluorescence Microscope (LSFM) to facilitate the visualization and quantification of developmental processes in living samples as well as the large-volume anatomical imaging of fixed, cleared tissues. The system will be housed in the Washington University Center for Cellular Imaging (WUCCI), a recently formed, shared technology resource at the School of Medicine. The Zeiss Lightsheet Z.1 microscope integrates an enclosed sealed-box imaging platform comprised of: i) a sample chamber and holder; ii) incubation, CO2, and microfluidics; iii) 405 nm, 488 nm, 561 nm, and 638 nm excitation lasers; iv) 5X dual illumination optics and v) multiple detection optics, including objective lenses optimized for cleared tissue imaging. This instrument will facilitate the long-term, time-lapse imaging of developing organisms and cultured organoids, as well as whole, fixed cleared tissues at subcellular resolution. This approach offers researchers several key advantages over conventional fluorescence microscopy modalities, including increased sample penetration; faster image acquisition rates, enhanced signal-to-noise ratios and significantly reduced illumination exposure, which results in minimal photobleaching and sample toxicity. Six investigators from five different departments at the School of Medicine will make use of this advanced imaging platform to study a wide-range of ongoing NIH-funded basic and translational research programs aimed at understanding vertebrate gastrulation, pathogenesis of heart failure, melanoma progression, kidney regeneration and the development of gastric cancers. This state-of-the-art imaging platform will complement the existing fluorescence imaging resources in the WUCCI by filling a current void in live organism imaging (allowing for multi-day time course, dynamic live imaging experiments) and whole, fixed cleared tissue imaging using approaches such as CLARITY. While the instrument has been configured to meet the specific needs of the major user group, we thoroughly expect it to impact many other projects as well. 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 and their combined leadership brings over 40 years of 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, the institution provided an initial investment of over $7M and dedicated 6,500 square feet of specifically designed lab space. In support of this S10 grant application, the institution will also commit $100,000 ($20,000 per year for five years) to ensure the long-term success of this instrument (see supporting letters from Drs. Perlmutter, Piston and Bonni and Solnica-Krezel).
Investigating developmental mechanisms and how they become dysfunctional in disease states is greatly augmented by the ability to visualize cellular and macromolecular behavior in living tissues. This proposal seeks to acquire a Lightsheet Fluorescence Microscope, (LSFM), a state-of-the-art imaging system that will facilitate the long-term, minimally phototoxic, three-dimensional imaging of living specimens such as zebrafish and cultured organoids with high spatial and temporal resolution. This platform will significantly enhance ongoing NIH-funded basic and translational research programs aimed at understanding vertebrate gastrulation, pathogenesis of heart failure, melanoma progression, stem cell biology and kidney regeneration and the development of gastric cancers.