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      Simultaneous protection of tissue physicochemical properties using polyfunctional crosslinkers


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          Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named ‘Stabilization under Harsh conditions via Intramolecular Epoxide Linkages to prevent Degradation’ (SHIELD), which uses a flexible polyepoxide to form controlled intra- and intermolecular crosslink with biomolecules. SHIELD preserved protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three dimensional (3D) phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.

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          Most cited references51

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          The green fluorescent protein.

          R Tsien (1998)
          In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
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            A mesoscale connectome of the mouse brain.

            Comprehensive knowledge of the brain's wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.
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              Interneuron cell types are fit to function.

              Understanding brain circuits begins with an appreciation of their component parts - the cells. Although GABAergic interneurons are a minority population within the brain, they are crucial for the control of inhibition. Determining the diversity of these interneurons has been a central goal of neurobiologists, but this amazing cell type has so far defied a generalized classification system. Interneuron complexity within the telencephalon could be simplified by viewing them as elaborations of a much more finite group of developmentally specified cardinal classes that become further specialized as they mature. Our perspective emphasizes that the ultimate goal is to dispense with classification criteria and directly define interneuron types by function.

                Author and article information

                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                6 October 2018
                17 December 2018
                17 June 2019
                : 10.1038/nbt.4281
                [1 ]Institute for Medical Engineering and Science
                [2 ]Picower Institute for Learning and Memory
                [3 ]Department of Chemical Engineering
                [4 ]Department of Brain and Cognitive Sciences
                [5 ]Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA
                [6 ]Broad Institute of Harvard University and MIT, Cambridge, MA, USA
                [7 ]Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
                [8 ]Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
                [9 ]Program in Cellular and Molecular Medicine, Boston Children’s Hospital and Harvard Medical School
                [10 ]Department of Mechanical and Automation Engineering, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
                [11 ]C.S. Kubik Laboratory for Neuropathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
                Author notes


                Y.-G.P., C.H.S., R.C., D.H.Y., and K.C. designed the study and wrote the paper with input from other authors. Y.-G.P. led development of SHIELD-SWITCH and SHIELD-MAP methods. C.H.S. and R.C. designed and performed the FISH experiments and protein analysis. R.C. and M.M. performed the fluorescence protein experiments. Y.-G.P., C.H.S., and M.M. performed the immunoreactivity experiments. Y.-G.P. and C.H.S. characterized physical properties of SHIELD tissue and performed axon tracing. D.H.Y. carried out active clearing and staining. D.H.Y. performed 3D phenotyping of the biopsy samples. G.T.D. and C.H.S. developed the SHIELD protocol for postmortem human brain tissues and performed immunostaining and imaging of the human tissues. T.K. performed the tissue expansion analysis. N.B.E. conducted light-sheet microscope imaging. H.C.O. and W.T. helped sample preparation. H.C. built temporally focused line-scanning microscope. M.C.T. and H.L.P. provided purified GFP. X.J. and T.R.G. provided tumor tissues. S.-C.C. and J.W. developed the oscillating blade microtome. M.P.F provided human brain tissues and validated the human brain data. H.J.K and H.W.Q. performed molecular simulation. V.L. and B.K.L. provided the virus, the virus labeled tissues, and helpful discussion. K.C. supervised all aspects of the work.

                [] Correspondence should be addressed to K.C. ( khchung@ 123456mit.edu ).

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