Researchers Achieve Targeted Control of Hundreds of Neurons Using Two-photon Optogenetic Stimulation

Researchers are increasingly focused on precisely controlling brain activity, and a new advance in two-photon optogenetic stimulation offers unprecedented possibilities for both understanding and interfacing with the nervous system. Riichiro Hira and Yoshikazu Isomura, from Tokyo Medical and Dental University, detail the technical development of this technique, which allows scientists to activate hundreds of targeted neurons simultaneously with remarkable speed and flexibility. Unlike traditional methods, two-photon optogenetics minimises unwanted activation of surrounding cells, offering a far more refined approach to studying neural circuits and behaviour. This work highlights the progression of key strategies, including spiral scanning and advanced holographic techniques, and explores how these technologies are paving the way for sophisticated brain-machine interfaces with transformative potential.

Two-Photon Microscopy and Neural Imaging Techniques

Research in neuroscience increasingly relies on advanced imaging and manipulation techniques to understand brain function. Two-photon microscopy stands out as a dominant technology, enabling researchers to visualize and control neural activity with unprecedented precision. Complementary techniques, such as mesoscopy, which provides large field of view imaging at subcellular resolution, and three-dimensional imaging methods, are expanding the scope of investigation. Optogenetics, a technique using light to control neurons, is frequently combined with two-photon microscopy to create closed-loop experiments where neural activity can be both observed and manipulated.

Researchers are also refining opsins, the light-sensitive proteins used in optogenetics, to improve their speed and specificity. Alongside these core technologies, scientists are developing sophisticated computational tools for image processing, data analysis, and holographic reconstruction. These tools are essential for interpreting the complex data generated by these experiments and for creating precise stimulation patterns. Current research focuses on understanding the organization and function of cortical circuits, particularly those involved in sensory processing, motor control, and decision-making.

Scientists are also investigating fundamental principles of neural dynamics, such as how circuits encode information and how the balance of excitation and inhibition shapes brain activity. Closed-loop experiments and brain-computer interfaces are emerging as powerful tools for interacting with neural circuits and studying learning. Beyond basic research, these technologies are showing promise in therapeutic applications, such as restoring vision through optogenetic therapy. Deep brain imaging is also benefiting from innovations like transparent graphene arrays, which allow for recording cellular activity at greater depths. Key trends in the field include miniaturization of probes for recording and stimulating neural activity, combining multiple imaging techniques for a more complete picture of brain function, and developing large-scale data analysis tools to handle the massive datasets generated by these experiments. Ultimately, the goal is to translate these technologies into clinical applications that can treat neurological disorders and improve human health.

Two-Photon Optogenetics Enables Single-Cell Precision

Researchers now achieve precise control over neuronal activity at the single-cell level using two-photon optogenetics, a significant advancement over traditional methods. Conventional techniques like electrical stimulation activate numerous surrounding neurons, lacking the targeted precision needed for detailed circuit analysis. While whole-cell patch clamping offers precision, it is limited to stimulating only a few neurons at a time. Two-photon optogenetics overcomes these limitations by harnessing the nonlinear properties of two-photon absorption, allowing researchers to stimulate precisely defined groups of neurons without spatial restrictions on the expression of light-sensitive proteins.

This technique utilizes near-infrared lasers to drive opsins, light-sensitive proteins, through two-photon absorption, confining excitation to a minuscule focal volume deep within brain tissue. Scientists shape this focal region to selectively stimulate specific neuronal populations, employing techniques such as spiral scanning, temporal focusing, and three-dimensional computer-generated holography. These methods allow for precise control over the timing and location of stimulation. Researchers envision integrating two-photon calcium imaging to monitor brain activity in real-time, feeding this data into artificial intelligence algorithms, and then using the AI to determine which combination of cells should be stimulated next via two-photon optogenetics. This creates a closed-loop system where brain activity and AI-driven stimulation interact dynamically, promising to transform brain-machine interfaces and potentially augment cognitive function.

Precise Neuronal Control via Two-Photon Optogenetics

Recent advances in two-photon optogenetics now enable precise, targeted modulation of neuronal activity with single-cell resolution, offering significant improvements over traditional electrophysiological methods and one-photon optogenetics. The technology allows for the simultaneous stimulation of hundreds of neurons with rapidly reconfigurable patterns, providing powerful tools for investigating neural circuits and behavior. Several strategies have emerged to optimize this approach, including spiral scanning, temporal focusing, and three-dimensional computer-generated holography, often used in combination to achieve precise control. Spiral scanning effectively stimulates cells at a rate of roughly one cell per second, while temporal focusing improves axial resolution by stretching and recompressing laser pulses, enabling near-instantaneous activation or inhibition of single neurons.

Holographic techniques further enhance control by generating holographic patterns to simultaneously excite specific groups of neurons. Recent studies demonstrate the ability to stimulate approximately 10 to 50 cells concurrently, activating a high percentage of targeted cells with precise timing. These advancements are paving the way for sophisticated brain-machine interfaces and a deeper understanding of the neural basis of behavior, offering unprecedented opportunities to investigate the complex workings of the brain.

Precise Neuronal Control via Two-Photon Optogenetics

Recent advances in two-photon optogenetics now enable precise, targeted modulation of neuronal activity with single-cell resolution, offering significant improvements over traditional electrophysiological methods and one-photon optogenetics. The technology allows for the simultaneous stimulation of hundreds of neurons with rapidly reconfigurable patterns, providing powerful tools for investigating neural circuits and behavior. Several strategies have emerged to optimize this approach, including spiral scanning, temporal focusing, and three-dimensional computer-generated holography, often used in combination to achieve precise control. The development of these methods has also spurred innovation in related technologies, such as multiplexed temporally focused computer-generated holography and the use of acousto-optic deflectors to achieve sub-millisecond stimulation updates. While current methods demonstrate considerable promise, challenges remain in controlling spot size and shape with certain techniques, and large-scale three-dimensional stimulation using acousto-optic deflectors remains a complex undertaking. Future research may focus on overcoming these limitations and integrating these technologies into brain-machine interfaces, though careful consideration of experimental design and methodological assumptions will be crucial for interpreting results and drawing firm conclusions about brain function.