The biology of multicellular organisms is coordinated across multiple size scales, from the sub-nanoscale 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 four-fold 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. Off-the-shelf antibodies can be used for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, with our experiments demonstrating 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.
An interesting profile has emerged from the results of this study, suggesting that certain psychological characteristics such as aggression, self-control, and narcissistic personality traits may predispose some individuals to become addicted to online games. This result will deepen our understanding of the "at-risk" population for online game addiction and provide basic information that can contribute to developing a prevention program for people who are addicted to online games.
SUMMARY 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.
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.
Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected, suggesting that the entire range of dorso-ventral identities can be generated within continuous neuroepithelia. Consistent with this, we demonstrate the presence of forebrain-organizing centers that express secreted growth factors, which may be involved in dorso-ventral patterning within organoids. Furthermore, we demonstrate the timed generation of neurons with mature morphologies, as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides critical methodology and quality criteria for phenotypic analysis of brain organoids, and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro.
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