Although confocal laser scanning microscopy has revolutionized the imaging of biological materials, its destructive properties impose limitations on the study of living tissues. Also, scattering and absorption in thick preparations of brain tissue limit the imaging depth of confocal techniques. While multiphoton microscopy promises to solve these problems, probe photobleaching within the focal volume of multiphoton excitation remains and issue, as probes and instruments that are truly optimized for this technique remain commercially unavailable. In response to NIH PA 99-007, """"""""Probes and Instruments for Micro-imaging the Brain,"""""""" Microcosm, Inc. proposes to introduce a new class of probes for two-photon microscopy. Based on metal-ligand charge transfer (MLCT) complexes, these probes will significantly improve two-photon imaging of thick, living brain preparations by providing enhanced sensitivity, reduced toxicity, greater phostostability, and specificity to a wide range of analytes. Additionally, we propose a modular, multifunctional system for use with existing commercial multiphoton microscopes which provides new experimental capabilities needed for the study of living brain and nervous tissues. Our system will be designed to perform two-photon optimized, truly localized uncaging of calcium and neurotransmitters within living brain preparation, while observing the temporal and spatial evolution of the resulting responses.
Currently, progress in multiphoton imaging of the brain is limited by commercial availability of good probes that enhance image quality, are photostable and non-toxic, and display specificity to analytes controlling brain functions. The proposed novel two-photon microscopy probes by Microcosm meet all of these requirements. This new class of probes, based on MLCT complexes, is not yet commercialized. The potential market for these probes is related to academic, pharmaceutical and medical research and diagnostics. The major drivers affecting the probe chemistry market are demands for: minimized costs of diagnostics, better quality and more sensitive imaging, real time imaging, and specific assays based on imaging. The proposed technologies can be applied to any complex biological tissue. The proposed hardware systems are commercially unavailable. A non-descanned interface is offered by Bio-Rad, but is sub- optimal. Our upgrades will enable new types of experiments needed in neuroscience and in other biomedical research. We expect that the multifunctional modual upgrades will be marketed world wide to pharmaceutical, industrial, hospital and university research labs.
