The proposed program will develop and refine a platform for in-vivo "Interventional Microscopy". The vision is to create an in-vivo microscope that can not just observe living cells in their natural environment, but directly interact with them. To achieve this they will implement photo-manipulation techniques more commonly applied to in-vitro studies, and translate them for use in-vivo in small animals. By refining experimental paradigms and demonstrating the efficacy of Interventional Microscopy techniques, they will introduce these potentially transformative new tools to a broad audience of biomedical researchers. To achieve these goals, they will start by developing a combined two-photon microscopy and photomanipulation instrument, based around an existing home-built in-vivo two-photon system. The design incorporates two independently focused and shaped beams from two different pulsed lasers, and will be operated by custom-written software. They will then begin developing and optimizing in-vivo techniques for using photoactivable compounds, as well as exploring optical trapping and particle-manipulation techniques in-vivo. To achieve, this they will develop and optimize procedures for in-vivo loading of photomanipulatable substances, as well as developing calibration techniques and controls to ensure that lightactivation in-vivo is reliable and well controlled. They plan to apply these techniques to their ongoing in-vivo studies of neurovascular coupling; the interrelation between blood flow and neuronal activity in the brain. Interventional Microscopy techniques will allow them to selectively manipulate the cells and vessels of the living brain, allowing in depth characterization of fundamental chemical and cellular mechanisms.
