(LIVE IMAGING CORE) The Live Imaging Core provides training in the use of, supervised access to, and maintenance of state-of-the- art imaging equipment, as well as the continued development of instrumentation and analysis methods. The three major equipment components of this Core, custom-built light sheet fluorescence microscopes, instrumentation for high throughput sample handling for light sheet microscopy, and tools for low- magnification fluorescence imaging are central to achieving the aims of the Research Projects. The technical expertise of this Core will ensure that all Project research is carried out on instrumentation that pushes the current boundaries for visualization of microbes within the zebrafish intestine. We have already demonstrated the ability to visualize larval zebrafish intestinal microbes with single-bacterium resolution, watching bacterial growth and competition and extracting quantitative information from the resulting images; these capabilities are requisite for our proposed Project experiments. Because our instrumentation is custom designed and built, it allows maximum flexibility for experimenters, including use of multiple fluorescent probes, and integration of custom-written software to control various hardware in ways tailored to the proposed experiments. In addition, image analysis code is also achieved by custom-developed software that makes use of semi- automated methods and machine-learning-based classification objects; ongoing work aims to further optimize these approaches. Our custom developed high throughput methods address the challenge of imaging large numbers of specimens and we are developing low-magnification ?macroscope? methods that employ simple yet versatile cameras and filters to rapidly assess abundances of fluorescent organisms over wide fields of view. This Core will continue to design and refine instrumentation. At the same time Core personnel will train Program Project research personnel in use of this instrumentation, and work with them to design experiments that maximize efficiency and productivity. Achieving the aims of this Core will significantly enhance the ability to carry out experiments described in the Research Projects.
