Highspeed Image Sensor and Microscopy Methods for Resolving Millisecond Scale Neural Dynamics in Freely Moving Mice and Zebrafish

Summary ): The application of functional fluorescence imaging to study neural circuit mechanisms in freely behaving animals has become a cornerstone approach in neuroethology. Recently, new fluorescence indicators, such as genetically encoded voltage (GEVI) indicators and new calcium indicators (GCaMP8), have achieved kinetics of milliseconds that can resolve activities as fast as single spikes. However, the current imaging systems used in different model animals have yet to attain the necessary resolution, speed, and signal-to-noise ratio (SNR) to image these indicators at sufficient speed (>1kHz) to resolve spiking events and the temporal coding of neuron ensembles, while accommodating unrestrained behaviors. The key challenges of high-speed fluorescence imaging in freely behaving animals, such as the spatial and temporal resolution trade-offs and the SNR requirements, are fundamentally linked to the camera image sensors. We have pioneered a novel CMOS image sensor with pixel-wise programmable exposures (“PE-CMOS”) to address these fundamental limitations. The PE-CMOS permits independent exposure at each pixel. This feature allows versatile pixel configurations to (1) enhance spatial-temporal resolution at sampling physiological signal while reducing system power and noise levels, (2) boost signal SNR in high-speed imaging with limited pixel exposure, and (3) accurately track cells’ position during brain movement caused by unconstrained behavior, and eliminating motion-induced artifacts. We will develop new image sensors based on the PE-CMOS architecture that will outperform the state-of-the-art image sensors. These are (1) a 600 x 800 pixels sensor with 1 kHz temporal resolution for fluorescence micro-endoscope in freely moving mice. (2) a 600 x 1300 pixel sensor with 11 kHz frame rate and an equivalent 50 kHz max temporal resolution for volumetric imaging of GEVI activity from the whole brain of a freely behaving zebrafish. We will demonstrate these sensors in applications to imaging GCaMP8 and GEVIs at high temporal resolutions while allowing for naturalistic behavior in mice and larval zebrafish — two widely used model systems in neuroscience research. Specifically, we will demonstrate (1) recording and decoding place cell replays at millisecond resolution and capture the spiking activity of fast-spiking interneurons, a task unattainable with current systems limited to frame rates below 60 - 100 Hz, and (2) we will also incorporate the new sensor into our recently demonstrated light-sheet microscopy system to allow whole-brain GEVI imaging of larval zebrafish at > 200 Hz temporal resolution during free tail movement. Achieving these goals will demonstrate the technology’s efficacy at resolving millisecond scale temporal coding structure in cell-type-specific neuron ensembles. It will further establish its applicability in other mammals and small animals commonly used in neuroscience research.