Miniature two-photon microscope for ultra-high-throughput calcium imaging in freely moving animals

The brain, an intricate network of billions of neurons, serves as the central hub for processing information and controlling complex behaviors. High-level cognitive functions—such as decision-making, social interaction, and navigation—emerge from the coordinated activity of large populations of neurons with distinct firing patterns and connections. However, our understanding of cognition has been significantly limited by the inability to simultaneously monitor the activity of large populations of neurons during animals' complex behaviors. Two-photon (2P) microscopy has become one of the most powerful techniques for large-scale imaging of neuronal activity in behaving animals. A key limitation of this technology is that traditional 2P microscopes are large, requiring animals to be head-fixed under the objective lens for imaging, much like human subjects must be immobilized for MR or CT scanning. To overcome this constraint, researchers have developed miniature 2P microscopes that can be mounted on freely moving animals, enabling the study of naturalistic behaviors. Despite significant technological advancements, existing miniature 2P microscopes are limited in throughput, with the typical number of neurons recorded simultaneously remaining at the hundreds level—only a small fraction of the total neurons in a typical brain microcircuit. Our project aims to revolutionize this field by developing a next-generation miniature 2P microscope. This system integrates several technological innovations that, when combined, increase the capacity to record neuronal activity from a few hundred to thousands of neurons—a potential 10-fold improvement over the current state of the art. If successful, this breakthrough will enable an unprecedented scale of neural recording in freely moving animals, providing transformative insights into the fundamental computational principles underlying high-level cognitive functions.

To uncover the neuronal mechanisms underlying complex cognitive functions, neuroscientists need to understand how information is encoded in populations of neurons. This requires measuring large-scale neuronal activity at cellular resolution in animals exhibiting natural behaviors. The current state-of-the-art technologies are silicon probes and miniaturized two-photon microscopes, or 2P miniscopes. 2P miniscopes offer significant benefits over silicon probes in terms of spatial resolution and the ability to record from the same neuronal population over extended periods. However, the recording capacity of 2P miniscopes is currently limited to hundreds of neurons, which falls short of the thousands of neurons needed to decipher the neurocomputational mechanisms within brain microcircuits. To bridge this gap, a pivotal development direction for this technology is to substantially increase the number of neurons that can be simultaneously recorded. In this project, I aim to push these boundaries by creating a new 2P miniscope (MINI10K) by upgrading most core components and introducing new designs to record, simultaneously, thousands of neurons in freely moving mice, increasing the neuron recording capacity by an order of magnitude compared to existing systems. I will demonstrate the MINI10K’s capability by measuring the spatial organization and population dynamics of various spatial cell types in a large anatomical area of the entorhinal cortex which is a critical step toward understanding the mechanism of cognitive map formation in the mammalian brain. The project is transformative in that it will enable us to explore population coding with unprecedented throughput and without behavioral restrictions, paving the way for studying the nseuronal mechanisms of cognitive functions at the network level.