Project Summary/Abstract Nonlinear optical (NLO) microscopy techniques have become important tools of inquiry for understanding both tissue biology and tissue pathology. Other than more conventional confocal fluorescence microscopy approaches, NLO microscopy enables label-free probing of tissue structures and components, at depths beyond what can be achieved with standard optical imaging techniques. NLO microscopy has become the method of choice for studying glycolysis and lipid metabolism a wide variety of tissues, studying myelin degeneration in nervous tissues, detecting migrating melanocytes in skin, mapping disease-induced changes to the extracellular matrix, and more. Novel advances in NLO microscopy are intimately linked to new scientific inquiries and discoveries in tissue biology. Since the 1990s, the Beckman Laser Institute (BLI) has played a leading role in developing NLO imaging technologies and applying these methods to solving outstanding problems in biology and biomedicine. To continue its pioneering role in advancing NLO imaging techniques, through this proposal the BLI is requesting a replacement of a laser-scanning NLO microscope, an ailing 12-year old user instrument. While this microscope has served more than 160 users, its vendor no longer services the instrument because of age, and its capabilities are incompatible with the evolving imaging needs of our user base. The requested replacement is a Leica SP8 multiphoton microscope, which is configured for high- resolution, meso-scale tissue imaging based on a wide variety of NLO contrast mechanisms: two- photon excited fluorescence (TPEF), second-harmonic generation (SHG) and third-harmonic generation (THG). In addition, we have worked with Leica engineers to enable imaging based on coherent anti-Stokes Raman scattering (CARS), a modality never before offered in combination with other femtosecond NLO modalities on a commercial laser-scanning microscope. The merger of all these NLO techniques in one instrument makes it possible to perform label-free imaging of lipids, protein density, carbohydrates, nucleic acids, collagen, NADH, elastin, melanin and more. Equipped with five sensitive detectors, fluorescence lifetime detection technology, resonant scanners, rapid mosaic-style image acquisition, enhanced spectral tuning of excitation and detection windows and an upright configuration with an open sample staging area, this unique instrument offers the advanced tissue imaging capabilities needed to propel the science of our user base into the next decade.