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

Comparison of local reactions to oxaliplatin infusions by peripheral versus central venous administration

Purpose Oxaliplatin, a platinum-type alkylator, is not classified as a vesicant but can cause local reactions when infused by peripheral vein. Providence Cancer Center, like other institutions, deferred the venous administration method to clinical judgment incorporating patient preference. Patient experience was evaluated for oxaliplatin-related local reactions by peripheral or central venous administration. Methods A retrospective review of the electronic medical record was performed of the period of January 2011 to March 2013 to identify patients who received oxaliplatin. Included were 59 patients who were given the option of either peripheral or central venous drug administration. The two patient groups (peripheral vein vs. central vein administration) were compared in terms of frequency and type of local reactions (redness/discoloration, swelling, numbness/cold, or pain/discomfort). Results Nineteen (63.3%) of the patients in the peripheral vein group experienced some type of local reaction compared to none in the central vein group ( p &lt; .0001). Pain was the most common local reaction, occurring in 17 (56.7%) patients in the peripheral group. Despite the occurrence of a local reaction, the majority of patients did not alter their initial choice of infusion method. Conclusion This is the first published report to characterize and quantify a single institution's experience with oxaliplatin-related local reactions. A significantly greater number of local reactions, particularly pain, occurred with the administration of oxaliplatin peripherally vs. centrally. This analysis impacted our institution's best practice for oxaliplatin infusions. </jats:sec

Scalable image processing techniques for quantitative analysis of volumetric biological images from light-sheet microscopy

AbstractHere we describe an image processing pipeline for quantitative analysis of terabyte-scale volumetric images of SHIELD-processed mouse brains imaged with light-sheet microscopy. The pipeline utilizes open-source packages for destriping, stitching, and atlas alignment that are optimized for parallel processing. The destriping step removes stripe artifacts, corrects uneven illumination, and offers over 100x speed improvements compared to previously reported algorithms. The stitching module builds upon Terastitcher to create a single volumetric image quickly from individual image stacks with parallel processing enabled by default. The atlas alignment module provides an interactive web-based interface that automatically calculates an initial alignment to a reference image which can be manually refined. The atlas alignment module also provides summary statistics of fluorescence for each brain region as well as region segmentations for visualization. The expected runtime of our pipeline on a whole mouse brain hemisphere is 1-2 d depending on the available computational resources and the dataset size.</jats:p

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

Multiscale 3D phenotyping of human cerebral organoids

AbstractBrain 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 ~ 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.</jats:p