Abstract This project describes the development of NeuroExM™, a highly innovative system for performing comprehensive spatial distribution analysis of populations of messenger RNAs (mRNAs) and proteins in tissue processed for expansion microscopy (ExM)). The groundbreaking technological advantage of ExM, which was recently developed by Dr. Edward S. Boyden (Dept. Biol. Engin., Media Lab and Dept. Brain Cognit. Sci., MIT, Cambridge, MA) and colleagues, is the ability to isotropically expand tissue and increase the size of the biological structures. This allows nanoscale-resolution, light-microscopic imaging of small objects that are too small to be resolved without expansion due to the diffraction limit of light. Among other benefits, ExM allows those small structures to be imaged with a wider range of microscopy techniques. Processing tissue for ExM also allows repeated hybridization (for investigations of mRNAs) and/or repeated antibody staining (for investigations of proteins) of the same tissue, combined with repeated microscopic imaging rounds. Each round yields adjacent, high-magnification, single field-of-view image stacks, consisting of at least one morphology reference channel showing neuronal sub-cellular structures (somas, axons, dendrites, dendritic spines, synapses) as well as one or several info channels showing mRNAs and/or proteins. Comprehensive analysis of the spatial distribution of populations of mRNAs and proteins in neurons in situ requires assembling the image stacks of all performed rounds into a single, seamless and aligned, three-dimensional (3D) ExM image, which is high-dimensional and can be several terabytes in size. However, this presents a number of computational challenges with respect to microscopy image registration, segmentation and analysis. The game-changing innovation in NeuroExM is the ability to perform all of these tasks without the need to have a computer scientist on staff to run the existing, individual lab-based software scripts developed for each step of this kind of complex analysis. This is made possible by implementing a number of significant technical innovations into NeuroExM. Based on pilot work performed in collaboration with the Boyden lab during preparation of this proposal, we are convinced that NeuroExM will make a significant impact on the field of neuroscience research. Specifically, the combination of ExM and NeuroExM will enable substantial advancements in research studies focusing on alterations in the spatial transcriptome and proteome of neurons associated with neurodevelopmental, neuropsychiatric, neurodegenerative and neurological disorders as well as in aging research and drug development. 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 feasibility of this novel technology by developing prototype software; work in Phase II will focus on creating the full functionality of NeuroExM for commercial release. We will perform extensive feasibility studies, product validation and usability studies of NeuroExM in close collaboration with the Boyden lab. A competing technology is not available.

Narrative Performing comprehensive spatial distribution analysis of populations of messenger RNAs and proteins in neurons in tissue processed for expansion microscopy holds the promise of profound progress in next-generation basic and translational neuroscience studies such as investigating the effects of aging, understanding neurodevelopmental, neuropsychiatric, neurodegenerative and neurological disorders and diseases, and drug discovery. Our proposed product will be a transformative technology, using a number of technical innovations that, for the first time, will enable researchers to perform these data-rich studies without the need to have a computer scientist on staff to run the existing, lab-based software developed for this kind of complex analysis. This system will allow researchers to make new discoveries based on new studies that are currently not feasible, ultimately providing the basis for developing novel treatments to prevent and fight complex brain diseases.