System for Volumetric 2-photon Imaging of Neuroactivity Using Light Beads Microscopy

Abstract This project aims to develop and commercialize the Volumetric Calcium Imaging 2-Photon Activity Microscope, vCAm™, a revolutionary new 2-photon microscope based on a technological breakthrough called Light Beads Microscopy (LBM) that was recently developed by Dr. Alipasha Vaziri and co-workers (Lab. Neurotechnol. Biophys., Rockefeller Univ., New York, NY). The game-changing innovation in the vCAm is the ability to perform unparalleled in vivo calcium imaging of individual neurons at cellular resolution nearly simultaneously in one or more cytoarchitectonic regions of the mouse cerebral cortex, and nearly simultaneously in 30 imaging planes each ~16 µm apart (i.e., up to a total depth of 500 µm, encompassing layers I-V) at a full-frame rate of at least 12 Hertz. These capabilities are crucial for ultimately correlating stimuli and/or behavioral states of an animal discretely, in a context-dependent manner, with the activity of all neurons in the brain of the animal that are involved in this process, which requires simultaneous recording of the activity of hundreds of thousands of neurons in a multi-regional and multi-layer manner. However, contemporary 2-photon microscopy suffers from a fundamental limitation. Neuroscience researchers need to record simultaneous interactions between the sensory, motor and visual regions of the brain, but it is difficult to capture the activity in such a broad volume of the brain without sacrificing resolution or speed. The LBM technology pushes the limits of imaging speed to the physical nature of fluorescence itself by eliminating the “dead time” between sequential laser pulses when no neuroactivity is recorded and at the same time the need for scanning. With this approach, the only limit to the rate at which samples can be recorded is the time that it takes the tags to fluoresce, meaning wide volumes of the brain can be recorded within the same time it would take a conventional two-photon microscope to capture a much smaller number of brain cells. Other technology, such as miniaturized 2-photon microscopes that can be carried on the head of freely moving rodents, functional magnetic resonance imaging, inserting electrodes into the brain, or fiber photometry do not fulfill this need. This project will improve upon the original LBM invention to create a commercial product for disseminating this important new technology. Based on pilot work performed at Dr. Vaziri's laboratory, it is clear that the vCAm will make a significant impact on the field of neuroscience research, including advancing studies focused on alterations in the circuitry of the central nervous system associated with neurodevelopmental, neuropsychiatric and neurodegenerative disorders. Ultimately, this will result in an improved basis for developing novel treatment strategies for a wide spectrum of complex brain diseases. In Phase I we will demonstrate the feasibility of this novel technology by developing prototype hardware and software; work in Phase II will focus on creating the full functionality of the vCAm for commercial release. We will perform extensive feasibility studies, product validation and usability studies of the vCAm in close collaboration with Dr. Vaziri. A competing technology is not commercially available.