Milling with Ultraviolet Excitation for Whole Organ Cellular-Level Phenotyping

PROJECT SUMMARY Many diseases, including neurodegenerative disease and cancer, induce complex changes to tissue microstructure. These include alterations in the microvasculature, cellular distribution, and neural connectivity. Researchers recognize that highly localized events, such as inflammation, lesions, and activated microglia, impact the formation of regional pathologies, such as heterogeneous tumors and neurodegenerative disease. These rare events are challenging to detect using traditional histology, given limitations in the field-of-view of optical microscopes and the lack of a three-dimensional histological context. The inability to comprehensively measure large-scale three-dimensional tissue microstructure leaves a critical gap in our understanding of neurodegenerative disease, limiting our ability to quantify new mechanisms for diagnosis and treatment. This proposal addresses this through commercialization of a high-throughput platform enabling multiplex three- dimensional imaging at rates over 1000X faster than existing microscopy. First, this proposal allows for the automation and transfer of published and patented imaging technology from the University of Houston to Swift Front, LLC. This novel imaging method, which we term milling with ultraviolet excitation (MUVE), enables acquisition of 400 million pixels per second with 3-channel molecular specificity, allowing three-dimensional sub- micrometer (≈1µm3) resolution imaging of 1cm3 samples in less than one hour. Samples are rapidly imaged through a combination of fast wide-field fluorescence imaging, deep-ultraviolet excitation, and serial ablation methods. This proposal will support Swift Frontandapos;s development of MUVE-compatible tissue labeling and processing protocols that will readily integrate with existing histology pipelines. State-of-the-art three-dimensional light-sheet imaging systems are constrained to 100μm thick samples, with the potential for 3-8mm depth with clearing and low numerical aperture objectives. Although these techniques have been proposed for samples approaching 1cm3, these methods are incompatible with routine phenotyping because they are slow, costly, and research lacks accessible computational tools for useful analysis. MUVE significantly increases acquisition speed by combining recent research in ultraviolet excitation with highly parallel data collection. This instrumentation also eliminates depth constraints inherent to optical microscopy by using new en bloc labeling techniques combined with tissue ablation. Our goal is to commercialize a platform that enables routine imaging, reconstruction, and analysis of large-scale phenotypes describing tissue microstructure and protein distribution. The availability of a platform for systematic tissue phenotyping and mapping will fundamentally impact biomedical research and education at the organ scale in the same way that satellite imagery, global positioning, and search algorithms have changed navigation.PROJECT NARRATIVE Comprehensive organ-scale three-dimensional tissue phenotypes are critical for understanding tissue disease progression and enable cellular-level quantification for drug discovery and treatment evaluation. Constructing deep phenotypic maps at such a massive scale requires the ability to image macroscopic samples, such as whole organs, at microscopic resolution. We propose instrumentation to provide radical advancement in the ability to map individual molecules of interest in human tissue at whole-organ scale and microscopic resolution. This research is relevant to NIH’s mission of developing new approaches critical for understanding tissue function and the mechanisms of disease.