Project Summary: Every cell and tissue biomedical research program requires microscopic visualization of research specimens. Some of this need can be addressed by routine widefield and confocal microscopy of relatively thin (< 50 um) tissue slices or tissue culture. However, biological understanding of molecular, cell and tissue-level dynamics has been uniquely enabled by the recent explosion of advanced, multi-parametric microscopy procedures for in vivo and ex vivo imaging. These procedures include multiphoton microscopy combined with harmonically generated signals (SHG) for imaging in thick (100s of microns) tissue and solid tumors, expressing multiple fluorescent proteins, as well as for deep (>1 mm), high resolution imaging of optically cleared tissues (e.g. Clarity, iDISCO) stained with multiple fluorescent markers. These techniques are now a required part of contemporary biomedical cell and tissue research. To be applied successfully, these methods require a versatile, advanced imaging system that incorporates fast scanning, multi-photon and 1-photon excitation at multiple wavelengths, high sensitivity and spectral selectivity over multiple detection channels, rapid imaging, and environmental control. The demands of these state-of-the-art microscopy methods for both specialized equipment and specialized expertise often outstrip the resources available to individual research laboratories. As such, these needs are best met with a shared instrument housed in a shared resource facility. For these reasons we are requesting funding to replace Stanford University's Cell Sciences Imaging Facility's 12- year-old in vivo multi-photon/confocal microscope (Leica SP5) with the new, state-of-the-art Stellaris 8 DIVE microscope. This combined multi-photon, confocal, in vivo, and ex vivo as well as in vitro imaging microscope will be a shared resource located in a well-established, multi-user light and electron microscopy facility: The Cell Sciences Imaging Facility (http://microscopy.stanford.edu). This facility is accessible to Stanford University's entire research community as well as surrounding biotech companies. The Stellaris 8 DIVE microscope will support research projects from 11 users, 10 of which are NIH funded. Their studies investigate critical functional and structural questions in a variety of model organisms and tissues and cover areas of biomedical research with implications for diverse aspects of human health and disease. These projects include: clonal fate and linage tracing of stem cells and tumor initiating cells, interrogation of the alveolar stem cell niche, molecular and cellular mechanisms of small cell lung and basal cell skin cancers development, characterization of skeletal stem cells in osteogenesis, characterization of the bone marrow microenvironment, gene therapy for inherited diseases and the biophysics of touch as well as characterizing. All these projects critically require advanced instrumentation for imaging live and fi...