This application is requesting support for the purchase of a confocal / multiphoton microscopy system that will be combined with an atomic force microscope (AFM) system. The combination of these two imaging modalities is unique and reversible involving only conversion of the microscope stages. Consequently, it can be operated with or without the AFM components satisfying the needs of users to use the microscope system with or without the AFM technology. Major uses of this technology will include studies of cell membrane integrins and their interactions with extracellular matrix proteins, focal contact and cytoskeletal proteins in response to mechanical loading at the level of a single focal contact, cell signaling responses in vascular and lymphatic smooth muscle, endothelial ceils in response to mechanical stimulation with the AFM, plasticity of three-dimensional smooth muscle organization in the wall of isolated and functional arterioles and lymphatic vessels, three-dimensional organizational characteristics of extracellular matrix proteins in the intact vascular wall, three-dimensional capillary tube networks in artificial extracellular matrices and spatial regulation of real time circadian gene expression in individual cells from suprachiasmatic nuclei (SCN) in vitro. The requested system will greatly facilitate our ability to perform repeated measurements over time at higher speeds and with less cellular and tissue damage. The unique capabilities of this hybrid imaging system that will allow us to perform these studies include: 1) limited biological damage to living cells and tissues; 2) in focus, optical sections of thick samples; 3) ability to view deep into slices and tissues; 4) the ability to image a variety of fluorescent molecules; and 5) increased ability to obtain three-dimensional data sets from cells and tissues and do this over time. This new technology will provide faculty, postdoctoral fellows and students with access to a unique state-of-the-art imaging facility and will facilitate the ongoing research of the major and minor users. ? ?
