Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.Simons Foundation. Postdoctoral FellowshipLife Sciences Research FoundationBurroughs Wellcome Fund (Career Award at the Scientific Interface)Searle Scholars ProgramMichael J. Fox Foundation for Parkinson's ResearchUnited States. Defense Advanced Research Projects AgencyNational Institutes of Health (U.S.) (1-U01-NS090473-01

Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues

The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.United States. National Institutes of Health (1-U01-NS090473-01

Scaling up 3D imaging, analysis, and culture of complex brain models

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, February, 2020Cataloged from the official PDF of thesis. "February 2020." Vita.Includes bibliographical references.The brain is the most complex human organ, containing components from the nanometer scale to the centimeter scale However, many experimental techniques in neuroscience have been optimized for small brain models This thesis summarizes a body of work aimed at scaling up 3D imaging, analysis, and tissue culture techniques for large-scale brain models We present a technique termed SWITCH that inhibits probe binding to allow for diffusion without the formation of a reaction front To improve imaging resolution, we present a tissue expansion technique called MAP that physically magnifies tissue samples for super-resolution imaging with conventional fluorescence microscopes Using these tools to achieve volumetric imaging of large-scale brain models generates petabyte-scale data, for which we present horizontally scalable image processing pipelines for analysis of intact mouse beams, marmoset bi am samples, and cerebral organoids The mouse brain pipeline allows region-based statistical analysis of protein expression and cell counts An efficient single-cell non-rigid coregistration algorithm for multiplexed volumetric fluorescence imaging based on matching corresponding nuclei between imaging founds is presented A multiscale phenotyping pipeline allows single-cell, cytoarchtectural, and morphological analyses to be combined into a hyperdimensional statistical analysis of cerebral organoids We use this pipeline to show phenotypic changes due to neurodevelopment, Zika virus infection, and changes in organoid culture protocols Current cerebral organoid cultures lack a vascular system and are limited by nutrient transport To address this issue in vitro, we fabricated synthetic vasculature by two-photon photopolymerization of polyethylene glycol-based resins Printed micro-vessels wee biocompatible, less than 100 [mu]m in outer diameter, and permeable to biomolecules through engineered pore structures Perfusion of vascularized cerebral organoids cultured for 30 days resulted neuronal differentiation as well as integration of the vascular network Future studies can use and build on these technical advances to further our understanding of the bi am through the use of large-scale brain models.by Justin M Swaney.Ph. D.Ph. D. Massachusetts Institute of Technology, Department of Chemical Engineerin

Multiscale 3D phenotyping of human cerebral organoids

Brain organoids grown from human pluripotent stem cells self-organize into cytoarchitectures resembling the developing human brain. These three-dimensional models offer an unprecedented opportunity to study human brain development and dysfunction. Characterization currently sacrifices spatial information for single-cell or histological analysis leaving whole-tissue analysis mostly unexplored. Here, we present the SCOUT pipeline for automated multiscale comparative analysis of intact cerebral organoids. Our integrated technology platform can rapidly clear, label, and image intact organoids. Algorithmic- and convolutional neural network-based image analysis extract hundreds of features characterizing molecular, cellular, spatial, cytoarchitectural, and organoid-wide properties from fluorescence microscopy datasets. Comprehensive analysis of 46 intact organoids and similar to 100 million cells reveals quantitative multiscale "phenotypes" for organoid development, culture protocols and Zika virus infection. SCOUT provides a much-needed framework for comparative analysis of emerging 3D in vitro models using fluorescence microscopy.11Nsciescopu

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study