Structural and molecular interrogation of intact biological systems.

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.

CLARITY for mapping the nervous system.

With potential relevance for brain-mapping work, hydrogel-based structures can now be built from within biological tissue to allow subsequent removal of lipids without mechanical disassembly of the tissue. This process creates a tissue-hydrogel hybrid that is physically stable, that preserves fine structure, proteins and nucleic acids, and that is permeable to both visible-spectrum photons and exogenous macromolecules. Here we highlight relevant challenges and opportunities of this approach, especially with regard to integration with complementary methodologies for brain-mapping studies.

Genetically targeted chemical assembly of polymers specifically localized extracellularly to surface membranes of living neurons.

Multicellular biological systems, particularly living neural networks, exhibit highly complex organization properties that pose difficulties for building cell-specific biocompatible interfaces. We previously developed an approach to genetically program cells to assemble structures that modify electrical properties of neurons in situ, opening up the possibility of building minimally invasive cell-specific structures and interfaces. However, the efficiency and biocompatibility of this approach were challenged by limited membrane targeting of the constructed materials. Here, we design a method for highly localized expression of enzymes targeted to the plasma membrane of primary neurons, with minimal intracellular retention. Next, we show that polymers synthesized in situ by this approach form dense extracellular clusters selectively on the targeted cell membrane and that neurons remain viable after polymerization. Last, we show generalizability of this method across a range of design strategies. This platform can be readily extended to incorporate a broad diversity of materials onto specific cell membranes within tissues and may further enable next-generation biological interfaces.

Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals.

The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.

Functional maturation of human neural stem cells in a 3D bioengineered brain model enriched with fetal brain-derived matrix.

Brain extracellular matrix (ECM) is often overlooked in vitro brain tissue models, despite its instructive roles during development. Using developmental stage-sourced brain ECM in reproducible 3D bioengineered culture systems, we demonstrate enhanced functional differentiation of human induced neural stem cells (hiNSCs) into healthy neurons and astrocytes. Particularly, fetal brain tissue-derived ECM supported long-term maintenance of differentiated neurons, demonstrated by morphology, gene expression and secretome profiling. Astrocytes were evident within the second month of differentiation, and reactive astrogliosis was inhibited in brain ECM-enriched cultures when compared to unsupplemented cultures. Functional maturation of the differentiated hiNSCs within fetal ECM-enriched cultures was confirmed by calcium signaling and spectral/cluster analysis. Additionally, the study identified native biochemical cues in decellularized ECM with notable comparisons between fetal and adult brain-derived ECMs. The development of novel brain-specific biomaterials for generating mature in vitro brain models provides an important path forward for interrogation of neuron-glia interactions.

Advanced CLARITY for rapid and high-resolution imaging of intact tissues.

CLARITY is a method for chemical transformation of intact biological tissues into a hydrogel-tissue hybrid, which becomes amenable to interrogation with light and macromolecular labels while retaining fine structure and native biological molecules. This emerging accessibility of information from large intact samples has created both new opportunities and new challenges. Here we describe protocols spanning multiple dimensions of the CLARITY workflow, ranging from simple, reliable and efficient lipid removal without electrophoretic instrumentation (passive CLARITY) to optimized objectives and integration with light-sheet optics (CLARITY-optimized light-sheet microscopy (COLM)) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution. The entire protocol takes from 7-28 d to complete for an adult mouse brain, including hydrogel embedding, full lipid removal, whole-brain antibody staining (which, if needed, accounts for 7-10 of the days), and whole-brain high-resolution imaging; timing within this window depends on the choice of lipid removal options, on the size of the tissue, and on the number and type of immunostaining rounds performed. This protocol has been successfully applied to the study of adult mouse, adult zebrafish and adult human brains, and it may find many other applications in the structural and molecular analysis of large assembled biological systems.