PROJECT SUMMARY: AIRYSCAN IMAGING TECHNOLOGY The objective of the new equipment is to connect highly productive investigators with state-of-the-art optical technology and applications that facilitate scientific progress and novel breakthroughs. The core user group of well-established cell biologists are members of the NIH-funded Mayo Clinic Center for Cell Signaling in Gas- troenterology (C-SiG). The Specific Aims of the grant are threefold. First, to provide a reliable, accessible, state-of-the-art imaging technology to a user group that will facilitate the study of GI cellular signaling cas- cades. Second, to educate and train individual members of the group in the use of sophisticated cellular imag- ing methods. Emphasis is placed on providing technical instruction and educating investigators on how such approaches can expand the scope and breadth of their independent research programs. Third, to develop and apply innovative fluorescence technologies, including biosensors and vital dyes, to study GI tissues and/or cells in health and disease pathogenesis. This work requires advanced imaging of highly dynamic and/or pho- tosensitive processes within cells while also defining the localization of molecular machinery components with high sensitivity and low phototoxicity. The newly developed Airyscan technology is a major breakthrough that enables this by: i) providing a perfect optical section with highest sensitivity due to its innovative detector array, ii) improving signal-to-noise and spatial resolution to best-in-class, allowing detection of interactions and pro- cesses never before seen, iii) increasing sensitivity to facilitate live imaging of photosensitive processes and structures for long periods (overnight) while minimizing photo damage, and iv) speeding up image acquisition rates for superior temporal resolution to capture highly dynamic events and transient interactions. The new technology will be a part of the C-SiG Optical Microscopy Core, for which confocal microscope utilization has more than tripled in the past 10 years. The Core also provides instruction, technical advice, data interpretation, and development of novel, innovative optical approaches to the study of signaling pathways in GI cells and tis- sues. These services cover a wide range of topics including: confocal microscopy coupled with computer- based 3-D image reconstruction; Fluorescence Resonance Energy Transfer (FRET) applications to measure dynamic protein-protein interactions; Fluorescence Recovery After Photobleaching (FRAP) that allows the quantitation of protein recruitment/turnover; Fluorescence Loss in Photobleaching (FLIP); microinjection of liv- ing cells; expression and use of fluorescence-based bioprobes that facilitates the study and localization of spe- cific signaling proteins and lipids; the development and application of specific photo-activatable caged- compounds that allow a precise temporal and spatial activation of desired signaling molecules i...