Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

Low-cost and scalable projected light-sheet microscopy for the high-resolution imaging of cleared tissue and living samples.

Light-sheet fluorescence microscopy (LSFM) is a widely used technique for imaging cleared tissue and living samples. However, high-performance LSFM systems are typically expensive and not easily scalable. Here we introduce a low-cost, scalable and versatile LSFM framework, which we named 'projected light-sheet microscopy' (pLSM), with high imaging performance and small device and computational footprints. We characterized the capabilities of pLSM, which repurposes readily available consumer-grade components, optimized optics, over-network control architecture and software-driven light-sheet modulation, by performing high-resolution mapping of cleared mouse brains and of post-mortem pathological human brain samples, and via the molecular phenotyping of brain and blood-vessel organoids derived from human induced pluripotent stem cells. We also report a method that leverages pLSM for the live imaging of the dynamics of sparsely labelled multi-layered bacterial pellicle biofilms at an air-liquid interface. pLSM can make high-resolution LSFM for biomedical applications more accessible, affordable and scalable.

Whole-brain mapping reveals the divergent impact of ketamine on the dopamine system.

Ketamine is a multifunctional drug with clinical applications as an anesthetic, pain management medication, and a fast-acting antidepressant. However, it is also recreationally abused for its dissociative effects. Recent studies in rodents are revealing the neuronal mechanisms mediating its actions, but the impact of prolonged exposure to ketamine on brain-wide networks remains less understood. Here, we develop a sub-cellular resolution whole-brain phenotyping approach and utilize it in male mice to show that repeated ketamine administration leads to a dose-dependent decrease in dopamine neurons in midbrain regions linked to behavioral states, alongside an increase in the hypothalamus. Additionally, diverse changes are observed in long-range innervations of the prefrontal cortex, striatum, and sensory areas. Furthermore, the data support a role for post-transcriptional regulation in enabling ketamine-induced neural plasticity. Through an unbiased, high-resolution whole-brain analysis, this study provides important insights into how chronic ketamine exposure reshapes brain-wide networks.

Whole-brain mapping reveals the divergent impact of ketamine on the dopamine system.

Ketamine is a multifunctional drug with clinical applications as an anesthetic, as a pain management medication and as a transformative fast-acting antidepressant. It is also abused as a recreational drug due to its dissociative property. Recent studies in rodents are revealing the neuronal mechanisms that mediate the complex actions of ketamine, however, its long-term impact due to prolonged exposure remains much less understood with profound scientific and clinical implications. Here, we develop and utilize a high-resolution whole-brain phenotyping approach to show that repeated ketamine administration leads to a dosage-dependent decrease of dopamine (DA) neurons in the behavior state-related midbrain regions and, conversely, an increase within the hypothalamus. Congruently, we show divergently altered innervations of prefrontal cortex, striatum, and sensory areas. Further, we present supporting data for the post-transcriptional regulation of ketamine-induced structural plasticity. Overall, through an unbiased whole-brain analysis, we reveal the divergent brain-wide impact of chronic ketamine exposure on the association and sensory pathways.

Scalable projected Light Sheet Microscopy for high-resolution imaging of living and cleared samples.

Light sheet fluorescence microscopy (LSFM) is a widely used imaging technique for living and large cleared samples. However, high-performance LSFM systems are often prohibitively expensive and not easily scalable for high-throughput applications. Here, we introduce a cost-effective, scalable, and versatile high-resolution imaging framework, called projected Light Sheet Microscopy (pLSM), which repurposes readily available off-the-shelf consumer-grade components and an over-the-network control architecture to achieve high-resolution imaging of living and cleared samples. We extensively characterize the pLSM framework and showcase its capabilities through high-resolution, multi-color imaging and quantitative analysis of mouse and post-mortem human brain samples cleared using various techniques. Moreover, we show the applicability of pLSM for high-throughput molecular phenotyping of human induced pluripotent cells (iPSC)-derived brain and vessel organoids. Additionally, we utilized pLSM for comprehensive live imaging of bacterial pellicle biofilms at the air-liquid interface, uncovering their intricate layered architecture and diverse cellular dynamics across different depths. Overall, the pLSM framework has the potential to further democratize LSFM by making high-resolution light sheet microscopy more accessible and scalable.

An in vitro model of neuronal ensembles.

Advances in 3D neuronal cultures, such as brain spheroids and organoids, are allowing unprecedented in vitro access to some of the molecular, cellular and developmental mechanisms underlying brain diseases. However, their efficacy in recapitulating brain network properties that encode brain function remains limited, thereby precluding development of effective in vitro models of complex brain disorders like schizophrenia. Here, we develop and characterize a Modular Neuronal Network (MoNNet) approach that recapitulates specific features of neuronal ensemble dynamics, segregated local-global network activities and a hierarchical modular organization. We utilized MoNNets for quantitative in vitro modelling of schizophrenia-related network dysfunctions caused by highly penetrant mutations in SETD1A and 22q11.2 risk loci. Furthermore, we demonstrate its utility for drug discovery by performing pharmacological rescue of alterations in neuronal ensembles stability and global network synchrony. MoNNets allow in vitro modelling of brain diseases for investigating the underlying neuronal network mechanisms and systematic drug discovery.