The focus of this 3 year proposal is developing a much needed microfluidic platform for long-term, high resolution optical imaging and recording of stimulated brain activity in living animals. Existing optical systems are limited by 1) requirements of relatively intense excitation light that causes photobleaching and phototoxicity that limits the duration of the experiment and hinders stimulating the neuronal circuit under investigation or 2) by the incompatability of optical systems that can operate at lower intensities with standard microfluidic stimulation methods. The central aim of this proposal is to develop microfluidic devices using optical index-matched materials compatible with a low intensity microscopy method, "selective plane illumination microscopy (SPIM)." Preliminary results show feasibility of hydrogel-based systems to record neural responses in C elegans for hours, as well as compatibility with simultaneous optogenetic (pulsed visible light) neural activation and readout. The proposed system will for the first time enable simultaneous chemical and optical stimulation and perturbation of brain circuits under investigation, with multiple neurons monitored for activity over several hours. The methods developed will impact the broader neuroscience community, in which neural imaging is a critical method for developmental, structural, and functional brain studies. Materials and hardware, including microfluidic systems, will be made accessible to the research and commercial community. Broader impact is also achieved through a comprehensive educational plan including innovative curricula in advanced biomedical imaging focused on neuroscience applications and outreach activities to increase involvement of STEM-underrepresented students and local communities, through exciting, hands-on modules for summer programs and scientific demonstrations.
Sensation, memory, and behaviors are encoded in dynamic electrochemical patterns within neurons of the brain. Recent advances in fast three-3D microscopy have enabled the optical imaging of activity in large numbers of neurons at once, in some cases nearly the entire brain of an organism. Such systems promise to revolutionize the study of neural circuit regulation, compared with sparse single-neuron recordings that do not capture neural dynamics elsewhere in the circuit. However, current confocal and structured illumination systems are limited by their requirements of relatively intense excitation light, causing photobleaching and phototoxicity that limits the duration of an experiment, and by difficulty in stimulating the neuronal circuit under investigation. While light sheet or selective plane illumination microscopy (SPIM) captures more emission light and therefore operates at lower excitation intensity, optical requirements are incompatible with standard microfluidic stimulation methods. Therefore, there exists an urgent need for SPIM-compatible microfluidic stimulation methods to enable long-term, high resolution recording of stimulated brain activity in living animals. The central aim of this proposal is to develop microfluidic devices using optical index-matched materials compatible with SPIM. Preliminary results show feasibility of hydrogel-based systems to record neural responses in C elegans for hours, as well as compatibility with simultaneous optogenetic neural activation and readout. Specific objectives are: 1) development of diSPIM-compatible sample immobilization and microfluidic stimulation, 2) multi-neuronal imaging of sensory-stimulated brain circuits to observe and study sensory feedback, and 3) multi-neuronal imaging of optogenetically-stimulated brain circuits to observe the effect of reversible circuit perturbations on ensemble neural activity in the same animal. Innovations of the propose include: 1) identification of hydrogel encapsulants compatible with diSPIM that immobilize living animals but maintain organism health and function; 2) microfluidic designs, including hydrogel-glass or hydrogel-silicone hybrids, that deliver precise chemical concentrations; 3) identification of sensory neurons detecting novel chemical stimuli; 4) identification of sensory feedback and study of its regulation via candidate genetic mutants; 5) simultaneous optogenetic and chemical stimulation while monitoring multiple sensory and interneurons, to observe neural responses during dynamic circuit perturbation. The end result will be a new configuration of the dual-view inverted (diSPIM) system and protocols suitable for the embedding of cells and small organisms to record high-resolution, isotropic, fluorescent 3-D volumetric images for long time periods during and after chemical and/or optogenetic stimulation. The studies planned will improve the understanding of circuit computation in C. elegans when stimulated with natural sensory stimuli (e.g., chemicals) and by arbitrary optogenetic stimulation within a compact and well-defined neural circuit. The methods developed will impact the broader neuroscience community, in which neural imaging is a critical method for developmental, structural, and functional brain studies. Materials and hardware, including microfluidic systems, will be made accessible to the research and commercial community. Integrated with the proposed research is a comprehensive educational program toward training and educating the next generation of interdisciplinary scientists, particularly biologist-engineers, including: 1) innovative curricula in advanced biomedical imaging with focus on neuroscience applications, 2) mentoring undergraduate and graduate students through research and engineering design projects, and 3) outreach to increase involvement of STEM-underrepresented students and local communities, through exciting, hands-on modules for summer programs and scientific demonstrations.
