Three-dimensional random access acousto-optical scanning two-photon microscope

Summary This NIH grant proposal seeks funding to acquire a cutting-edge Femtonics Atlas microscope at Baylor College of Medicine (BCM), with a specific focus on its applications for neuroscience research. The Atlas will enable rapid, high-resolution imaging of activity-dependent fluorescence in vitro and in vivo using calcium, voltage, or neurotransmitter reporters. The Atlas combines two-photon excitation with acousto- optical deflectors (AOD) scanning, allowing fast recordings of fluorescence from arbitrary locations or shapes in a 3D volume without moving parts. Over the past two decades, traditional two-photon laser-scanning microscopy (2PLSM) has significantly advanced our understanding of neuronal function and structure due to its ability to image activity deep in scattering tissue. However, standard 2PLSM is limited by single-plane scanning, which is often not compatible with the geometry of target structures such as dendrites or astrocytes. When combined with methods for fast Z focusing, 2PLSM can scan multiple planes, but at the expense of reduced temporal resolution. The Femtonics Atlas overcomes these limitations by rapidly pointing the laser beam to any point within a 3D volume, enabling high-speed, random-access imaging that is crucial for recording fast neuronal activity with the latest GCaMP8 calcium sensors and with genetically-encoded voltage indicators (GEVIs) like JEDI-2P (developed at the St-Pierre lab here at BCM). The proposal highlights the unique advantages of the Atlas to support a diverse set of NIH-funded projects within the neuroscience department at BCM, with potential applications in other departments as well. These projects will benefit enormously from the ability to track activity in complex 3D structures of neurons and glia, to image sparsely-labeled cells with high temporal resolution, and to deliver precise two-photon optogenetic stimulation in parallel with imaging. With a group of nearly a dozen enthusiastic neuroscientists looking forward to applying the Femtonics Atlas’s capabilities to their research, bringing this microscope to Houston has the potential to significantly advance our understanding of brain function and disease.