Activity-sensitive fluorescent indicators and microscopy have proven valuable tools for measuring neuronal activity, but most forms of cellular microscopy can produce images of neurons only near the brain surface and generally only after removal of tissues overlying the brain surface, such as bone. Many neurons in neocortex are out of reach of cellular microscopy. Here we propose to optimize 3-photon (3P) excitation fluorescence imaging, a form of cellular microscopy, to enable deeper imaging into the brain.
Our aims are to acquire cellular information from deep cells in flies, mice, cats and macaques. After optimizing 3P microscopy, we will facilitate the duplication of our microscope and procedures in other neuroscience laboratories. We will provide open-access, detailed information on our microscope and experimental procedures in terminology that?s comprehensible to neuroscientists. In addition, we will work with one or more microscope manufacturers to ensure commercial availability of our microscope design, enabling many neuroscience laboratories to use 3P excitation to image deep into the brain with less invasive preparations, repeatedly over many days, weeks or months. To achieve our broad aims, we will pursue five specific aims: ? Specific aim 1: Further optimize fast 3P excitation. ? Specific aim 2: Record calcium signals from central motor circuits in intact, behaving Drosophila melanogaster. ? Specific aim 3: Study sensory information processing in mouse neocortex and spatial memory in mouse hippocampus. ? Specific aim 4: Study visual information processing in deep layers of cat and macaque cortex. ? Specific aim 5: Share methods and facilitate availability of a commercial fast 3P microscope.
In this proposal we will further develop a 3-photon excitation fluorescence microscopy, for optical access to tissues that are inaccessible with current imaging techniques. Our experiments will enable long-term imaging in laboratory animals, permitting neuroscientists to answer previously intractable questions about brain function in health and disease. Our technique also takes the field a step closer to being able to perform cellular-resolution experiments in human patients.
