We request funds to purchase a Perkin Elmer Opera LX microscope for fully automated, point-scanning confocal, high-throughput microplate imaging. Conventional inverted and confocal microscopes, plate readers and high-throughput scanners at Vanderbilt University cannot support automated long-term, high-resolution obser- vation of dynamic cellular events such as chemokinesis, chemotaxis and haptotaxis, cell division and differen- tiation, organelle function, receptor and protein trafficking, and apoptosis in large numbers of individual cells. There are no instruments at Vanderbilt comparable to the Opera LX. Key features offered by the Opera LX are the microlens spinning disk confocal microscope with a 10x air objective and 20x and 60x automated water objectives, three lasers and a tunable UV source, IR autofocus, two cameras that enable simultaneous two-color imaging and sequential multicolor imaging, and rapid insertion and XYZ positioning of plates. The dual-core processor allows simultaneous image acquisition and sophisticated processing by multiple users. Most importantly, the Opera can observe in parallel the changes in cellular behavior over hours to days for hundreds of separate populations of live cells maintained in 96 or 384 well plates or custom-fabricated microfluidic devices. This instrument will support a highly collaborative group of 12 major and 14 minor users from 13 departments in the College of Arts &Science and the Schools of Medicine and Engineering. Ongoing, NIH-funded projects by major users that will benefit include research on serotonin protein transporter trafficking;cellular behavior including chemotaxis, haptotaxis, paracrine signaling, and endothelial attachment in structured microenviron- ments;identification and characterization of genome maintenance proteins, the genes that regulate PIP3 pro- duction, and the role of ENOX1 in the morphogenesis of endothelial cells exposed to radiation;and the ability of stem cells to mediate cardiac ischemic injury. Minor projects include receptor trafficking, signaling dynamics, cardiac sarcomere turnover, cell-matrix interactions, and bacterial and viral infectivity. For each project, the requested instrumentation will have an immediate and substantial impact by providing unique new measurement capabilities of the biological phenomena under study. Acquisition and operation of this microscope by the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) will guarantee user training/support, 24 hour/day 7 day/week access, and instrument maintenance and repair. User fees will cover staff support, service and repair. Other reseachers will be able to use available time. VIIBRE has an outstanding re- cord in developing and delivering advanced microfluidic and microfabrication technologies, electronic instru- mentation, training, and user facilities to over 200 students and faculty at Vanderbilt. Public Health Rele- vance: The proposed state-of-the-art instrument will give researchers a new capability to study processes in development, wound repair, aging, cancer and heart failure that involve complex dynamic intracellular and extracellular signaling networks, thereby improving our ability to maintain health and diagnose and treat disease.
