RESUMO
We developed entangled link-augmented stretchable tissue-hydrogel (ELAST), a technology that transforms tissues into elastic hydrogels to enhance macromolecular accessibility and mechanical stability simultaneously. ELASTicized tissues are highly stretchable and compressible, which enables reversible shape transformation and faster delivery of probes into intact tissue specimens via mechanical thinning. This universal platform may facilitate rapid and scalable molecular phenotyping of large-scale biological systems, such as human organs.
Assuntos
Hidrogéis/química , Coloração e Rotulagem/métodos , Engenharia Tecidual/métodos , Acrilamida/química , Animais , Fenômenos Biomecânicos , Materiais Biomiméticos/química , Bioimpressão , Córtex Cerebral/química , Reagentes de Ligações Cruzadas/química , Módulo de Elasticidade , Hipocampo/química , Humanos , Teste de Materiais , Camundongos , Estresse Mecânico , Resistência à TraçãoRESUMO
Understanding cellular architectures and their connectivity is essential for interrogating system function and dysfunction. However, we lack technologies for mapping the multiscale details of individual cells and their connectivity in the human organ-scale system. We developed a platform that simultaneously extracts spatial, molecular, morphological, and connectivity information of individual cells from the same human brain. The platform includes three core elements: a vibrating microtome for ultraprecision slicing of large-scale tissues without losing cellular connectivity (MEGAtome), a polymer hydrogel-based tissue processing technology for multiplexed multiscale imaging of human organ-scale tissues (mELAST), and a computational pipeline for reconstructing three-dimensional connectivity across multiple brain slabs (UNSLICE). We applied this platform for analyzing human Alzheimer's disease pathology at multiple scales and demonstrating scalable neural connectivity mapping in the human brain.
Assuntos
Doença de Alzheimer , Encéfalo , Imagem Molecular , Humanos , Doença de Alzheimer/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Imagem Molecular/métodos , Fenótipo , Hidrogéis/química , ConectomaRESUMO
The complex connectivity of the mammalian brain underlies its function, but understanding how interconnected brain regions interact in neural processing remains a formidable challenge. Here we address this problem by introducing a genetic probe that permits selective functional imaging of distributed neural populations defined by viral labeling techniques. The probe is an engineered enzyme that transduces cytosolic calcium dynamics of probe-expressing cells into localized hemodynamic responses that can be specifically visualized by functional magnetic resonance imaging. Using a viral vector that undergoes retrograde transport, we apply the probe to characterize a brain-wide network of presynaptic inputs to the striatum activated in a deep brain stimulation paradigm in rats. The results reveal engagement of surprisingly diverse projection sources and inform an integrated model of striatal function relevant to reward behavior and therapeutic neurostimulation approaches. Our work thus establishes a strategy for mechanistic analysis of multiregional neural systems in the mammalian brain.
Assuntos
Mapeamento Encefálico , Imageamento por Ressonância Magnética , Animais , Encéfalo/fisiologia , Corpo Estriado , Imageamento por Ressonância Magnética/métodos , Mamíferos , Ratos , RecompensaRESUMO
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 ~ 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.
Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Organoides/citologia , Organoides/crescimento & desenvolvimento , Encéfalo/citologia , Diferenciação Celular , Humanos , Processamento de Imagem Assistida por Computador , Células-Tronco Pluripotentes Induzidas/citologia , Microscopia de Fluorescência/métodos , Rede Nervosa/diagnóstico por imagem , Neurônios/citologia , Fenótipo , Células-Tronco Pluripotentes/citologiaRESUMO
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 cross-link with biomolecules. SHIELD preserves 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 phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.