LEICA TCSSP2RS TWO-PHOTON MICROSCOPE FOR IN VIVO IMAGING

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This subproject is one of many research subprojects utilizing the resources provided by a Shared Instrumentation Grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the grant, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): The recent development of two-photon microscopy has made it possible to see within tissues to depths approaching 0.5mm. This makes it feasible to investigate the movements and interactions of cells in living organs. This technology clearly has an important future in immunology. The Intravital two-photon imaging system requested is the Leica Microsystems TCS SP2 RS (resonance scanning) confocal microscope, with infrared laser. This is the state-of-the-art for fast, deep-tissue imaging of motile cells, which is what is required to image cell-cell interactions in tissues such as the lymphoid organs. The user group have a strong background in imaging techniques. They are interested in a wide range of subjects under the broad definition of immunology. There is a particular interest in T cell activation in response to pathogens, cell-cell interactions, tumor growth, and in differentiation. These processes occur in specialized compartments in lymphoid tissues and are therefore not amenable to analysis in vivo by other microscopic techniques. The Leica TCS SP2 RS instrument is based on an upright microscope to allow easy mounting of bulk tissues or living animals, for intravital microscopy. The instrument's MaiTai laser is software tunable to allow relatively fast wavelength changes (within several seconds). The scanning head with resonant galvanometer is a critical component of the system. Resonant scanning is currently the fastest method, approaching video frame rates. The instrument has internal detectors with adjustable bandpass filters. Software controlled motorized slits are used to pass a range of wavelengths split by the prism. This approach is superior to using interference filters because it allows full freedom in optimization of each channel for given fluorophore and has better pass-through efficiency. In addition, 2-channel external (non-descanned) detectors allow maximum utilization of scattered fluorescence. Overall, this instrument will allow the user group to radically improve their ability to perform experiments on understanding the immune system in development and disease.