Two-photon Light Field with Neuro-active Sensing for Fast Volumetric Neural Microcircuit Readout

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Underlying our every sensation, thought, memory, decision and action are 100 billion neurons communicating through trillions of electrical impulses each second. Over the past century, neuroscientists have explored brain function on primarily two scales, that of single neurons (i.e., by impaling them with electrodes) and that of entire brain regions (i.e. with electroencephalogram, EEG, and functional magnetic resonance imaging, fMRI). However, between these two scales lies a large knowledge gap surrounding how neurons interact in networks to process and store information, form memories and generate actions. Over the past 10 years, geneticists have developed methods to control and read out brain cell activity with light. They can render neurons sensitive to light to activate or silence them when illuminated with certain wavelengths. In addition, neurons can be made to "glow" or become more fluorescent when active. These "optogenetic" tools make it possible to connect single-neuron properties (i.e., through electrode studies) with functions evolving on the population level (through fMRI and EEG). To achieve this, optical engineers must first overcome a key challenge: the mammalian brain severely scatters and distorts light, resulting in blurry images and thus confusion about which neuron is active. Here we propose to overcome this limitation by utilizing the "optogenetic" ability to activate individual neurons with light in rapid succession. Specifically, we will activate each neuron throughout a brain volume in turn to determine each one's "signature"; that is, the blurry, distorted light pattern it generates when active. We will then use this collection of activity signatures to rapidly and precisely determine which neuron activates and when during subsequent spontaneous activity. We will implement this "collection" approach with a three-dimensional (3D) imaging strategy called "light field." While traditional imaging captures focused images for objects lying in a single plane, "light field" captures perspectives from different angles within a single shapshot. The "light field" approach thus enables us to track neuronal activity simultaneously throughout a volume a brain tissue rather than within a single plane. This novel combination of "light field" imaging with active sensing will significantly increase the speed (10-fold) with which we can track the activity of single neurons throughout a volume. In the near future, development of faster, more sensitive cameras and sensors could increase our instrument's volume capture rates to 100-fold compared to the current state-of-the-art. Moreover, here we will, for the first time, implement "light field imaging" in "two-photon" mode. "Two-photon" is a method to excite fluorescence that is used widely in biomedical research. In contrast to the blue/green wavelengths previously used with "light field," "two-photon" utilizes near-infrared wavelengths that are far less scattered than blue and green, enabling researchers to image deep in scattering tissues. Our new two-photon light field instrument will decrease distortion and thus enable us to image deeper into the brain.By combining targeted neural activation with 3D light-field imaging, we will overcome a key barrier to understanding how neurons interact in networks. With our new instrument, neuroscientists will at last be able to collect data on how neurons work together to process and store information, make decisions and effectuate actions. A detailed understanding of these network-level processes will inform the design of new therapies for neuronal diseases and disorders, such as Alzheimer's, in which these functions are compromised.