In vivo imaging of cellular and molecular structures in the intact brain provides a powerful tool for wideranging investigations in normal physiology, or in experimental models of disease processes. We have recently developed methods using a light microscope-based technique, multiphoton microscopy, to image microscopic structures in the brains of living transgenic mice over periods of months. Multiphoton microscopy utilizes a near-infrared laser for excitation of fluorophores deep within scattering tissue, with high spatial and temporal resolution. The spatial resolution of this imaging technique is about 1micrometer, several orders of magnitude better than other in vivo techniques, like PET, or MRI. In this application, we propose to develop new techniques that will provide important in rive readouts for biological imaging. This research will also lay the groundwork for development of contrast reagents suitable for use in human brain imaging with PET or MRI. We will develop, in Aim 1, techniques for high-resolution, in vivo imaging of structural reporters in the brain. We will investigate procedures to image individual neurons and microglia with high spatial resolution in the intact brain. In Aim 2, we propose to develop imaging techniques that exploit functional reporters in these living cells in the brain. Development of these molecular imaging techniques will build upon techniques accomplished in Aim 1. We have been using an experimental, transgenic mouse model of Alzheimer's disease that develops senile similar to those found in patients with Alzheimer's disease (AD). Our imaging techniques have allowed us to image the senile plaques in vivo in these mice with high spatial resolution. We will apply our new imaging techniques to this mouse model and address important questions that will provide insight into the pathophysiology of this disease. Our current techniques, however, rely on invasive procedures to gain access to the brain for imaging. In Aim 3, we will develop new techniques for non-invasive, in rive detection of senile plaques. New techniques using nearinfrared contrast reagents, and IR-sensitive detectors will allow non-invasive detection of plaques in the intact animal, and may also lead to clinically relevant diagnostic procedures for AD patients. In summary, the proposals outlined in this application will lead to generally applicable new techniques for cellular and molecular imaging in the intact brain.