Acquisition of a Leica Stellaris 5 Upright Confocal Microscope for In Vivo Imaging Applications - Project Summary/Abstract The purpose of this shared S10 instrumentation grant application is the purchase of a Leica upright Stellaris 5 confocal microscope to replace an obsolete, end-of-life Leica SP-5 confocal microscope dedicated for live animal imaging. The main aim is to continue to provide high-resolution intravital confocal imaging capabilities to the University of Miami UM research community by upgrading and replacing the existing instrument (now 18 years old) that has reached the end of its life and can no longer be repaired or serviced. The requested microscope fills a critical need and will support the ongoing efforts of the NIH-funded research of 9 Major User groups representing 5 Departments at the University of Miami (UM). Other than the microscope we are replacing, there are no other available, open access microscopes at UM configured for live animal imaging studies that meet the needs of the existing and future User Groups. The replacement Leica Stellaris 5 system is configured with a white light laser (WLL) having an excitation range from 495 to 790 and a detection range up to 850 nm. It also has a 405 nm laser for near-UV excitation. The extended excitation wavelengths provided by the WLL allows for additional fluorescent reporters to be imaged simultaneously in a single experiment, thereby providing additional information compared with the older technology that was limited to the detection of 4 or 5 fluorescent reporters at best. The Stellaris 5 system enabling the multiplexing of fluorescent reporters in a single experiment also reduces the utilization of research animals consistently with the 3Rs of animal research (Replacement, Reduction, Refinement). The microscope is equipped with a 5x and a long- distance 10X dry objects and with apochromatic water-immersion objectives (16x, 25x, and 40x) with high numerical aperture and large free working distances that are essential for intravital imaging. The upright configuration of the objective lenses is required for proper access to the live animal tissue to be imaged and positioning of the objectives to regions of interest. The upright configuration and long working distance objectives are necessary to accommodate the anesthesia apparatus and stereotaxic head-holder, which require marked clearance from the microscope body and objectives mounting turret to allow positioning of the animal on the stage and under the objective. The instrument will be housed and maintained at the open access Analytical Imaging Core Facility (AICF), where the entire UM community has access to instrumentation on a fee-for-service basis. The existing confocal microscopes in the AICF and other imaging facilities at the University, other than the obsolete Leica SP-5 system, allow for high precision state-of-art imaging of ex vivo tissues and live cells in culture vessels; however, the inverted configuration of those systems and the objective lenses they are equipped with are not amenable for live animal imaging, for the reasons elaborated above. Therefore, the replacement of the existing upright SP-5 system that reached its end-of-life is critically needed.
