Project Summary The ability to collect high resolution volumetric datasets of whole organs is arguably most applicable to the neuroscience field. Individual neurons on the micron scale comprise circuits that extend across large brain territories over millimeters apart; thus, perturbations of neuronal circuits in models of neurological, psychiatric or brain injury conditions can become evident only by assessing volumetric datasets spanning the micro-, meso- and macro-scales. We are requesting funds to purchase a TissueVision TissueCyte 1600FC serial two-photon tomography (STPT) microscope to enable high-throughput, high resolution automated imaging of whole, uncleared rodent brains or spinal cord segments by NIMH/NIH supported investigators at UT Southwestern Medical Center in Dallas, TX. STPT is a block face volumetric imaging modality in which two-photon mosaic tile imaging is alternated repeatedly with vibrotome sectioning to obtain full 3D volumes of the entire brain or spinal cord segment. The instrument will be housed in and maintained by the Whole Brain Microscopy Facility (WBMF) and will be available to all investigators on campus as well as external users. This instrument will complement and expand the capacity of two existing STPT systems within the facility (TissueVision TissueCyte 1000 - both 9 years old) which are heavily used (1100 total samples imaged since 2014; projected total of 168 samples for CY23) and lack many features of the updated TissueCyte 1600FC. Our existing TC1000 systems have a single tunable Ti:Sapphire excitation laser and are configured for detection of three emission channels (blue, green and red), which greatly limits their range of detectable fluorophores and multiplexing capability. The new TC1600FC system offers a dual laser line system, four detection channels and the innovative robotic “SlicePlacer” module to automatically mount the sections onto slides after imaging for correlative staining. Our existing TC1000s cannot accommodate imaging of far-red fluorophores, multiplex imaging of fluorophores with widely separated excitation spectra, ratiometric imaging of inherent autofluorescent signals, label free nonlinear microscopy methods such as second and third harmonic generation (SHG and THG), or automatic mounting of sections. The WBMF has a full time computational scientist on staff and robust, custom developed registration and analysis pipelines to accommodate quantification of STPT whole brain and cord datasets. There are no other STPT microscopes available on the UT Southwestern campus or in the state of Texas to our knowledge. The WBMF has a broad user base, currently serving 237 active clients from 98 individual laboratories across 31 basic science and clinical departments with a wide variety of project foci. Since the installation of the current TissueCyte 1000 instruments in 2014, we have supported STPT experiments from 41 individual investigators, the vast majority of which are neuroscience focused...