Confocal upgrade for intravital microscopy following vascular injury 6. Project summary The goal of this proposal is to modernize an existing shared instrument that has been used for the past 5 years to observe the hemostatic response to vascular injury in genetically engineered mice. Hemostasis refers to the rapid accumulation of circulating platelets and fibrin at a site of injury, thereby limiting blood loss. The unintended consequence of having a rapid response hemostatic mechanism is that inappropriate formation of platelet/fibrin clots is a primary contributor to heart attacks and strokes, particular in the setting of atherosclerosis. Although much has been learned about these events in vitro, it has only recently been possible to study them in vivo. Basic and clinical research in hemostasis and thrombosis research is a major focus at Penn Medicine and Children's Hospital of Philadelphia (CHOP). Five years ago, we assembled a (then) state of the art instrument that has enabled us to use digital Intravital fluorescent microscopy to observe events within the microcirculation. The original instrument has been put to good use in studies that range from testing new ideas about platelet activation to efforts to finding novel ways to cure hemophilia. It has been shared by members of the immediate community as well as by visiting investigators. When originally assembled, there was only one comparable instrument in the United States and even now there are few others. However, the original instrument included confocal capabilities whose usefulness proved to be limited by the relatively long times (>45 sec) required to capture an entire set of images. Better, faster confocal heads are now available and can be retrofitted to our existing microscope. The requested funds will be used to upgrade the confocal head, lasers and camera, and replace the wavelength changer with one that is much faster. The net effect will be to allow us to observe events in greater detail and with a greater sense of spatial relationships, bringing us back to the level of state of the art technology needed to answer biologically-important questions. We know that the new system will work as expected, because a similar instrument was recently completed in the Furie lab at Harvard. Although our specific biological questions differ, their experience is informative about the capabilities of the system. This proposal includes examples of the NIH-funded research that will benefit from the new instrument, as well as a management plan for its continued use. Economic impact: Penn Medicine and CHOP contribute substantially to the local economy. In 2008, they created and supported more than 54,000 jobs and $11 billion in regional economic activity. The current proposal will help us to maintain our edge in competing for grant support and create or retain at least 4 jobs at Penn plus more at Intelligent Imaging Innovations, our partner in developing and maintaining the instrument. 1

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biomedical Research Support Shared Instrumentation Grants (S10)
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Special Emphasis Panel (ZRG1-CB-J (31))
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Levy, Abraham
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University of Pennsylvania
Internal Medicine/Medicine
Schools of Medicine
United States
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Welsh, John D; Muthard, Ryan W; Stalker, Timothy J et al. (2016) A systems approach to hemostasis: 4. How hemostatic thrombi limit the loss of plasma-borne molecules from the microvasculature. Blood 127:1598-605
Ivanciu, L; Stalker, T J (2015) Spatiotemporal regulation of coagulation and platelet activation during the hemostatic response in vivo. J Thromb Haemost 13:1949-59
Welsh, John D; Stalker, Timothy J; Voronov, Roman et al. (2014) A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets. Blood 124:1808-15
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Ivanciu, Lacramioara; Krishnaswamy, Sriram; Camire, Rodney M (2014) New insights into the spatiotemporal localization of prothrombinase in vivo. Blood 124:1705-14
Stalker, Timothy J; Welsh, John D; Tomaiuolo, Maurizio et al. (2014) A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity. Blood 124:1824-31
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Stalker, Timothy J; Traxler, Elizabeth A; Wu, Jie et al. (2013) Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood 121:1875-85
Welsh, J D; Colace, T V; Muthard, R W et al. (2012) Platelet-targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse. J Thromb Haemost 10:2344-53