Multi-photon laser scanning microscopy is a powerful new tool to probe brain structure and function within the intact, living nervous system. For example, recent in vivo studies have shown that the anatomy and ionic behavior of neurons in the mammalian cortex can be probed with micrometer spatial resolution and millisecond temporal resolution. Yet formidable problems remain in the application of optical techniques to in vivo studies, particularly since light is strongly scattered by brain tissue so that it is difficult to resolve structures deep to the pial surface of cortex. Here we propose to advance multi-photon imaging techniques as a means to enable studies of brain homeostasis and neuronal and glial dynamics at the level of the middle and deep layers of mammalian cortex. Our technical program is focused on the optimization of pulsed lasers for deep imaging. We propose to construct a source that is tunable with center wavelengths from 525 nm to beyond the water absorption limit of approximately 1.3 mum, whose pulse shape is optimized for multi-photon absorption, and, most critically, whose trade-off between energy per pulse and repetition rate insures the greatest possible penetration depth into cortex while heating of the brain and possible photodynamic damage in minimized. The proposed improvements should extend the depth for diffraction limited imaging of brain structures from the current state-of-the-art of approximately 500 mum to over 1 mm. These improvements will be amenable to a broad range of extrinsic, intrinsic and genetically induced molecular indicators of brain function in vivo. Our research effort toward the study of cortical microcirculation provides a context for our technical development. Fine capillaries are ubiquitous throughout the depth of cortex and thus provide a test bed to readily explore issues of depth penetration and photodynamic damage. Our preliminary data shows that conventional two-photon laser scanning microscopy may be used to observe the flow of individual red blood cells in capillaries that lie in the superficial layers of cortex in rat. We will use our proposed advancements in penetration depth to map the spatial-, temporal-, and stimulus-dependence of changes in cerebral microcirculation throughout the entire depth of cortex as a means to address the microscopic connection between neuronal activity and local blood flow. The proposed advancement in the depth penetration of multi-photon methods will provide an essential tool for understanding the optical properties of normal and diseased tissues, and thus may substantially improve upon the effectiveness of two-photon diagnostic procedures and photodynamic therapies.
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