Inverted multi-photon confocal microscope - PROJECT SUMMARY The University of Colorado Anschutz medical campus requests funds to purchase a Zeiss LSM980 confocal/2- photon microscope system equipped with fluorescence lifetime imaging (FLIM). This is to replace an equivalent LSM780 confocal/2-photon FLIM system that was purchased in 2013 but is now aging and will very soon no longer be supported by the manufacturer. The LSM780 system we seek to replace represents the only inverted 2-photon microscope system available for use on campus. This system has received consistently high usage since its purchase (>185h/month in 2023, close to maximum capacity) by 149 principal investigators over the past 5 years, and it is critical to support NIH funded research across a wide range of disciplines. The proposed system consists of a Zeiss LSM980 confocal microscope system with spectral detector unit that is equipped for 2-photon excitation via coupling of a Ti;sapphire fs infra-red laser, and inclusion of non-descanned detectors. This system also includes a specialized Picoquant multiharp FLIM module that consists of fast detectors and high-speed photon counting card. This Zeiss LSM980 confocal/2- photon FLIM system will be housed within the institutional Advanced Light Microscopy Core (ALMC). The ALMC is well equipped to house the instrument, which includes in depth expertise with the technologies included via PhD-level staff. The ALMC also receives strong institutional support from the school of medicine via the Neurotechnology center (NTC). This includes -2000sq ft of space that houses 10 instruments, as well as salaries for 3 full-time professional staff and support to backstop any budget shortage to cover instrument service contracts. For the proposed system, institutional support in the shape of $155,000 is provided, as well as a recent purchase of a new Ti:saphhire laser. In choosing the proposed Zeiss LSM980 system, we tested 3 other microscope systems and quantified multiple aspects of the system performance (resolution, detection sensitivity, live sample handling), finding the Zeiss system showed superior performance. Similarly, we determined the proposed Picoquant FLIM module provided the most accurate lifetime measurements. Every component of the system is required by all major users (5) and either the majority of or all minor users (8). These users represent those performing the most cutting-edge microscopy in their NIH-funded research that includes 2-photon microscopy and FLIM and requiring the highest level of usage. These users demonstrate the broad range of microscopy use among the 149 principal investigators who are using the current aging system. Thus, replacement of the current Zeiss LSM780 confocal/2-photon FLIM system with the proposed Zeiss LSM980 confocal/2-photon FLIM system is urgently needed in order to maintain ongoing access to required multi-spectral confocal microscopy, 2-photon microscopy and FLIM, for NIH funded research.
