Collaborative Research: Two-photon DeepSpiral super-resolution imaging of in vivo molecular dynamics in deep tissue

To understand how the brain works, researchers want to investigate the tiniest parts within the brain's cells and tissues in living animals. However, most microscopes can only see clearly near the surface of the brain because light gets scattered and blurry as it goes deeper into the tissue. This means that important details, like how certain proteins and molecules are organized far below the surface, are hard to study. The project team plans to develop DeepSpiral, an innovative microscopy technique designed to solve this problem. This new type of single-molecule localization microscopy will herald an unprecedented era for cellular biology, particularly neuroscience, where a better understanding of the molecular mechanisms underlying brain functions such as learning is needed. Technology will combine multiphoton and single-molecule localization microscopies to achieve super-resolution at deep imaging depths. Thus, opening up new possibilities for understanding how the brain works and adapts, which could lead to discoveries about learning and memory. Though the project’s primary impact will be on understanding of the brain’s fundamental structural processes of learning, the proposed innovation on super-resolution at depth would enable novel measurements and applications in other biological systems, engineering and chemistry. The project team has an extensive track record of mentoring and training students, and they will expand these efforts in the proposed work on interdisciplinary training in fluorescence microscopy, neuroscience, and materials science, providing critical training and networking activities for young scientists to connect with industrial opportunities and needs. The innovation in tool development, computational modeling, and insights into the super-resolution processes of learning contribute significantly to developing and advancing the national priorities on biotechnology and innovation. The proposed DeepSpiral is an innovative super-resolution microscopy technique that would enable nanometer-scale imaging of molecular structures at depths up to 300 microns within living brain tissue that is far beyond the reach of current methods. DeepSpiral incorporates illumination with a dynamically structured beam at low excitation power to unlock super-resolution and minimize tissue heating, providing multi-photon excitation and incoherent fluorescence detection to image through thick scattering tissue. DeepSpiral will enable answering fundamental questions in neuroscience, such as how individual brain structures determine function and how timescales and relative locations of brain structures change in the learning process. The primary impact will be a new understanding of the fundamental structural processes of learning by the brain, but it will also have a significant impact in other fields of biology, besides applications in the field of solid-state systems such as layered computer chips or studying heterostructures of 2D materials. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.