Abstract To understand how the brain processes information and generates behaviors, we must record neural activity of three-dimensionally distributed circuits at millisecond timescales. While current optical imaging methods successfully reached imaging rates at kHz frame rates, they are still too slow to resolve the calcium dynamics of neurons throughout an entire volume or to track large populations of neurons in interconnected regions of brain in moving animals. The goal of this proposal is to overcome the limitations of optical imaging of the brain with a new two-photon microscopy technique enabling calcium imaging beyond 100,000 frames rates, which is 100x faster than currently possible. Our proposed two-photon line excitation array detection (2p-LEAD) imaging modality achieves hundreds of kHz framerates using a novel implementation of the fastest mode of ultrafast line- scanning with an acousto-optic deflector, and sensitive detection using a photomultiplier array. The sensitive, fast imaging can enable mapping of the flow in information in neural circuits at millisecond timescales. We recently developed the LEAD microscopy as the fastest fluorescence imaging modality and demonstrated the system’s high signal-to-noise ratio, despite scanning at an unprecedented speed of 0.8 million frames per second. These encouraging results and our preliminary results with 2p-LEAD ensure the success of our main goal in this proposal: to implement 2p-LEAD microscopy in vivo at the highest speeds possible without causing thermal damage to brain. In Aim 1, we will build the 2p-LEAD microscope using acousto-optic scanning to reach 125,000 frame per second which will enable probing 500 neurons within a volume of 240 x 200 x 300 cubic micron within 0.8 ms (1,250 Hz volumetric rates). We will test the microscope’s performance using agar phantoms with varying scattering properties mimicking the brain. Next, we will perform in vivo calcium imaging in the mouse brain to test heating, sensitivity, imaging depth, resolution, the effect of scattering on image blurring, and the systems capability for tracking neural activity in slightly moving samples. We will implement several strategies to reduce heating while using high laser power, such as an “on-off” imaging cycle and perfusion of a cooled immersion medium or cooling of the carotid artery, which will be performed carefully to match the heating effects from imaging. Our goal is to identify the parameter space (FOV, resolution, laser power, imaging depth) 2p-LEAD can operate for brain imaging. In Aim 2, we will implement a large area scanning to enable monitoring up to 10,000 neurons across 3 x 3 x 0.3 cubic millimeter volume at 30 Hz sampling rate. We will implement this new method for calcium imaging in the primary visual cortex of mice, which is an attractive model to study intercortical communication with two-photon imaging, as it consists of several distinct areas that are compactly laid out on the surface across a ~3 mm area. This unique experiment will allow us to quickly assess new questions about interareal connectivity.

Public Health Relevance Statement: Understanding how the brain processes information, which gives rise to behavior and is altered by neurological diseases, requires monitoring the brain at the speed of neuron activity. The proposed project, for the first time, will open the possibility for such studies by developing the fastest imaging method that will enable monitoring of neural activity at their firing speed.