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Studying chemosensory processing desires precise chemical cue presentation, behavioral response monitoring, and large-scale neuronal activity recording. Here we present Fish-on-Chips, a set of optofluidic tools for highly-controlled chemical delivery while simultaneously imaging behavioral outputs and whole-brain neuronal activities at cellular resolution in larval zebrafish. These include a fluidics-based swimming arena and an integrated microfluidics-light sheet fluorescence microscopy (µfluidics-LSFM) system, both of which utilize laminar fluid flows to achieve spatiotemporally precise chemical cue presentation. To demonstrate the strengths of the platform, we used the navigation arena to reveal binasal input-dependent behavioral strategies that larval zebrafish adopt to evade cadaverine, a death-associated odor. The µfluidics-LSFM system enables sequential presentation of odor stimuli to individual or both nasal cavities separated by only ~100 µm. This allowed us to uncover brainwide neural representations of cadaverine sensing and binasal input summation in the vertebrate model. Fish-on-Chips is readily generalizable and will empower the investigation of neural coding in the chemical senses.

Pharmacological reversal of brain aging is a long-sought yet challenging strategy for the prevention and treatment of age-related neurodegeneration, due to the diverse cell types and complex cellular pathways impacted by the aging process. Here, we report the genome-wide reversal of transcriptomic aging signatures in multiple major brain cell types, including glial and mural cells, by systemic glucagon-like peptide-1 receptor (GLP-1R) agonist (GLP-1RA) treatment. The age-related expression changes reversed by GLP-1RA encompass both shared and cell type-specific functional pathways that are implicated in aging and neurodegeneration. Concomitantly, Alzheimer’s disease (AD)-associated transcriptomic signature in microglia that arises from aging is reduced. These results show the feasibility of reversing brain aging by pharmacological means, provide mechanistic insights into the neurological benefits of GLP-1RAs, and imply that GLP-1R agonism may be a generally applicable pharmacological intervention for patients at risk of age-related neurodegeneration., Li, Chen, Vong, Zhao et al. show that glucagon-like peptide-1 receptor agonist reverses transcriptomic aging signatures in mouse glial and neurovascular cells. This study suggests that glucagon-like peptide-1 receptor agonists may be considered as a therapeutic option for patients at risk of age-related neurodegeneration.

We demonstrate second-harmonic generation (SHG) microscopy excited by the ∼890-nm light frequency-doubled from a 137-fs, 19.4-MHz, and 300-mW all-fiber mode-locked laser centered at 1780 nm. The mode-locking at the 1.7-µm window is realized by controlling the emission peak of the gain fiber, and uses the dispersion management technique to broaden the optical spectrum up to 30 nm. The spectrum is maintained during the amplification and the pulse is compressed by single-mode fibers. The SHG imaging performance is showcased on a mouse skull, leg, and tail. Two-photon fluorescence imaging is also demonstrated on C. elegans labeled with green and red fluorescent proteins. The frequency-doubled all-fiber laser system provides a compact and efficient tool for SHG and fluorescence microscopy.

Aims A variety of tissue clearing techniques have been developed to render intact tissue transparent. For thicker samples, additional partial tissue delipidation is required before immersion into the final refractive index (RI)‐matching solution, which alone is often inadequate to achieve full tissue transparency. However, it is difficult to determine a sufficient degree of tissue delipidation, excess of which can result in tissue distortion and protein loss. Here, we aim to develop a clearing strategy that allows better monitoring and more precise determination of delipidation progress. Methods We combined the detergent sodium dodecyl sulphate (SDS) with OPTIClear, a RI‐matching solution, to form a strategy termed Accurate delipidation with Optimal Clearing (Accu‐OptiClearing). Accu‐OptiClearing allows for a better preview of the final tissue transparency achieved when immersed in OPTIClear alone just before imaging. We assessed for the changes in clearing rate, protein loss, degree of tissue distortion, and preservation of antigens. Results Partial delipidation using Accu‐OptiClearing accelerated tissue clearing and better preserved tissue structure and antigens than delipidation with SDS alone. Despite achieving similar transparency in the final OPTIClear solution, more lipids were retained in samples cleared with Accu‐OptiClearing compared to SDS. Conclusions Combining the RI‐matching solution OPTIClear with detergents, Accu‐OptiClearing, can avoid excessive delipidation, leading to accelerated tissue clearing, less tissue damage and better preserved antigens., Our new strategy of using Accurate dilapidation with Optimal Clearing combines SDS, OPTIClear and refractive index‐matching solution. The new strategy provides fast, non‐swell/shrink, and no protein loss protocol for making the brain to be semi‐transparent. This opens a new avenue of fast pathological analysis of the brain without many thin tissue sectioning.

The molecular signatures of cells in the brain have been revealed in unprecedented detail, yet the ageing-associated genome-wide expression changes that may contribute to neurovascular dysfunction in neurodegenerative diseases remain elusive. Here, we report zonation-dependent transcriptomic changes in aged mouse brain endothelial cells (ECs), which prominently implicate altered immune/cytokine signaling in ECs of all vascular segments, and functional changes impacting the blood–brain barrier (BBB) and glucose/energy metabolism especially in capillary ECs (capECs). An overrepresentation of Alzheimer disease (AD) GWAS genes is evident among the human orthologs of the differentially expressed genes of aged capECs, while comparative analysis revealed a subset of concordantly downregulated, functionally important genes in human AD brains. Treatment with exenatide, a glucagon-like peptide-1 receptor agonist, strongly reverses aged mouse brain EC transcriptomic changes and BBB leakage, with associated attenuation of microglial priming. We thus revealed transcriptomic alterations underlying brain EC ageing that are complex yet pharmacologically reversible., Blood–brain barrier dysfunction occurs in ageing and in neurodegenerative diseases. Here, the authors use scRNA-seq to identify transcriptomic changes in endothelial cell subtypes in the aged mouse brain, some of which may generalize to human and can be reversed by treatment with a GLP-1R agonist.

Parallelized fluorescence imaging has been a long-standing pursuit that can address the unmet need for a comprehensive three-dimensional (3D) visualization of dynamical biological processes with minimal photodamage. However, the available approaches are limited to incomplete parallelization in only two dimensions or sparse sampling in three dimensions. We hereby develop a novel fluorescence imaging approach, called coded light-sheet array microscopy (CLAM), which allows complete parallelized 3D imaging without mechanical scanning. Harnessing the concept of an “infinity mirror”, CLAM generates a light-sheet array with controllable sheet density and degree of coherence. Thus, CLAM circumvents the common complications of multiple coherent light-sheet generation in terms of dedicated wavefront engineering and mechanical dithering/scanning. Moreover, the encoding of multiplexed optical sections in CLAM allows the synchronous capture of all sectioned images within the imaged volume. We demonstrate the utility of CLAM in different imaging scenarios, including a light-scattering medium, an optically cleared tissue, and microparticles in fluidic flow. CLAM can maximize the signal-to-noise ratio and the spatial duty cycle, and also provides a further reduction in photobleaching compared to the major scanning-based 3D imaging systems. The flexible implementation of CLAM regarding both hardware and software ensures compatibility with any light-sheet imaging modality and could thus be instrumental in a multitude of areas in biological research., Microscopy: parallel light sheets give a gentler view A new imaging technique called coded light-sheet array microscopy (CLAM) brings an improved ability to visualize biological samples in three dimensions while minimizing the damage. A research team, led by Kevin Tsia at the University of Hong Kong, developed the technique and demonstrated its use on a variety of biological imaging scenarios. CLAM is a new form of 3D fluorescence microscopy, which collects all the fluorescence signals from the entire illuminated volume of a sample in parallel. Its key innovation is the way it uses a concept called “infinity mirror” to generate an array of parallel slices of laser light (“light sheets”) for three-dimensional imaging without the need for repetitive scanning and with much gentler illumination than the state-of-the-art techniques. CLAM could be applied to existing light sheet fluorescence microscopes, with minimal system modifications.

Mounting evidence suggests that distinct microbial communities reside in tumors and play important roles in tumor physiology. Recently, a previous study profiled the composition and localization of intratumoral bacteria using 16S ribosomal DNA (rDNA) sequencing and histological visualization methods across seven tumor types, including human glioblastoma. However, their results based on traditional histological examinations should be further validated considering potential sources of contamination originating from sample collection and processing. Here, we aim to propose a three-dimensional (3D) in situ intratumoral microbiota visualization and quantification protocol avoiding surface contamination and provide a comprehensive histological investigation on local bacteria within human glioma samples. We develop a 3D quantitative in situ intratumoral microbiota imaging strategy, combining tissue clearing, immunofluorescent labeling, optical sectioning microscopy, and image processing, to visualize bacterial lipopolysaccharide (LPS) within gliomas in a direct, contaminant-free, and unambiguous manner. Through an automated statistical algorithm, reliable signals can be distinguished for further analysis of their sizes, distribution, and fluorescence intensities. In tandem, we also combined 2D images obtained from thin-section histological methods, including immunohistochemistry and fluorescence in situ hybridization, to provide comprehensive histological imaging for local bacterial components within human glioma samples. We have, for the first time, achieved 3D quantitative imaging of bacterial LPS colonized in gliomas in a contamination-free manner within human glioma samples. We also built the multiple histological evidence chain demonstrating the irregular shapes and sparse distribution of bacterial components within human glioma samples, mostly localized near nuclear membranes or in the intercellular space. This study provides favorable evidence for the presence of microbiota in human gliomas and provides information on the feature and distribution of bacterial components. The results, along with the integrated 3D quantitative intratumoral microbiota imaging method, are promising to provide insightful information into the direct interactions between the microbial community and the host in the tumor microenvironment.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV., Simeprevir, an HCV drug, suppresses SARS-CoV-2 replication by inhibiting two viral proteins and modulating host immune responses, thus providing novel insights in anti-CoV drug design.

Summary Optimizing navigational strategies in nature requires the detection and processing of survival-relevant cues. Our understanding of how animals utilise parallel inputs from paired sensory organs for this purpose and the underlying neural circuit mechanisms remain limited. Here we developed microfluidics-based behavioral and brainwide imaging platforms to study the neural integration of bilateral olfactory inputs and chemosensory avoidance in larval zebrafish. We show that larval zebrafish efficiently escape from cadaverine-carrying streams using undirected large turns, where both angular velocity and adaptation of turn duration exhibit bilateral olfactory input-dependence. In contrast, concomitant swim bout frequency modulation (i.e., klinokinesis) only requires unilateral input. Throughout the olfactory processing pathways, a distributed neural representation with a wide spectrum of ipsilateral-contralateral stimulus selectivity is maintained. Nonlinear sensory information gain with bilateral signal convergence is especially prominent in neurons weakly encoding unilateral cadaverine stimulus, and associated with stronger activation of sensorimotor neurons in the downstream brain regions. Collectively, these results provide insights into how the vertebrate model sums parallel input signals to guide navigational behavior.

Pharmacological reversal of brain aging is a long-sought yet challenging strategy for the prevention and treatment of age-related neurodegeneration, due to the diverse cell types and complex cellular pathways impacted by the aging process. Here, we report the genome-wide reversal of transcriptomic aging signatures in multiple major brain cell types, including glial and mural cells, by glucagon-like peptide-1 receptor (GLP-1R) agonist (GLP-1RA) treatment. The age-related expression changes reversed by GLP-1RA encompass both shared and cell type-specific functional pathways that are implicated in aging and neurodegeneration. Concomitantly, Alzheimer’s disease (AD)-associated transcriptomic signature in microglia that arises from aging is reduced. These results show the feasibility of reversing brain aging by pharmacological means, provide mechanistic insights into the neurological benefits of GLP-1RAs in diabetic patients, and imply that GLP-1R agonism may be a generally applicable pharmacological intervention for patients at risk of age-related neurodegeneration.

Due to the complexity and limited availability of human brain tissues, for decades, pathologists have sought to maximize information gained from individual samples, based on which (patho)physiological processes could be inferred. Recently, new understandings of chemical and physical properties of biological tissues and multiple chemical profiling have given rise to the development of scalable tissue clearing methods allowing superior optical clearing of across-the-scale samples. In the past decade, tissue clearing techniques, molecular labeling methods, advanced laser scanning microscopes, and data visualization and analysis have become commonplace. Combined, they have made 3D visualization of brain tissues with unprecedented resolution and depth widely accessible. To facilitate further advancements and applications, here we provide a critical appraisal of these techniques. We propose a classification system of current tissue clearing and expansion methods that allows users to judge the applicability of individual ones to their questions, followed by a review of the current progress in molecular labeling, optical imaging, and data processing to demonstrate the whole 3D imaging pipeline based on tissue clearing and downstream techniques for visualizing the brain. We also raise the path forward of tissue-clearing-based imaging technology, that is, integrating with state-of-the-art techniques, such as multiplexing protein imaging, in situ signal amplification, RNA detection and sequencing, super-resolution imaging techniques, multiomics studies, and deep learning, for drawing the complete atlas of the human brain and building a 3D pathology platform for central nervous system disorders.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients.One Sentence SummaryDiscovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

With advances in single-cell genomics, molecular signatures of cells comprising the brain vasculature are revealed in unprecedented detail1,2, yet the ageing-associated cell subtype transcriptomic changes which may contribute to neurovascular dysfunction in neurodegenerative diseases3–7 remain elusive. Here, we performed single-cell transcriptomic profiling of brain endothelial cells (EC) in young adult and aged mice to characterize their ageing-associated genome-wide expression changes. We identified zonation-dependent transcriptomic changes in aged brain EC subtypes, with capillary ECs exhibiting the most transcriptomic alterations. Pathway enrichment analysis revealed altered immune/cytokine signaling in ECs of all vascular segments, while functional changes impacting the blood-brain barrier (BBB) and glucose/energy metabolism were most prominently implicated in ECs of the capillary bed – the primary site where ECs and other neurovascular unit (NVU) cell types closely interact and coordinate to regulate BBB and cerebral blood flow in health and diseased conditions8–17. Furthermore, an overrepresentation of Alzheimer’s disease (AD)-associated genes identified from GWAS studies was evident among the human orthologs of differentially expressed genes of aged capillary ECs but not other EC subtypes. Importantly, for numerous EC-enriched differentially expressed genes with important functional roles at the BBB and/or association with AD, we found concordant expression changes in human aged or AD brains. Finally, we demonstrated that treatment with exenatide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, strongly reverses transcriptomic changes in ECs and largely reduces BBB leakage in the aged brain. Thus, our study provides insights into detailed transcriptomic alterations underlying brain EC ageing that are complex with subtype specificity yet amenable to pharmacological interventions.

In the original version of this Article, the concentration of boric acid buffer for the SDS clearing solution was given incorrectly as ‘1 M sodium borate’ and should have read ‘0.2 M boric acid’. Also, the composition of PBST incorrectly read ‘1% Triton X-100 (vol/vol) and 0.1% sodium azide (wt/vol)’ and should have read ‘0.1% Triton X-100 (vol/vol) and 0.01% sodium azide (wt/vol)’. Further, the pH of the OPTIClear solution was not stated, and should have read ‘with a pH between 7 to 8 adjusted with hydrochloric acid’. These errors have been corrected in both the PDF and HTML versions of the Article.

Previously, high-resolution three-dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide-based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel-embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA). The whole procedure is less time-consuming and less error-prone, and can be completed within a week, compared to conventional CLARITY protocols that may take weeks to complete. Here, the detailed stepwise procedures involved in the simplified CLARITY workflow are applied to the pancreas of wild-type and gene-knockout 6-week old mice expressing green fluorescent protein (GFP) under the mouse insulin 1 promoter (MIP-GFP). This technique could facilitate high-resolution, three-dimensional imaging of pancreatic islets and comparison between different mouse genotypes under different disease and treatment conditions. © 2017 by John Wiley & Sons, Inc.

Newly developed tissue clearing techniques can be used to render intact tissues transparent. When combined with fluorescent labeling technologies and optical sectioning microscopy, this allows visualization of fine structure in three dimensions. Gene-transfection techniques have proved very useful in visualizing cellular structures in animal models, but they are not applicable to human brain tissue. Here, we discuss the characteristics of an ideal chemical fluorescent probe for use in brain and other cleared tissues, and offer a comprehensive overview of currently available chemical probes. We describe their working principles and compare their performance with the goal of simplifying probe selection for neuropathologists and stimulating probe development by chemists. We propose several approaches for the development of innovative chemical labeling methods which, when combined with tissue clearing, have the potential to revolutionize how we study the structure and function of the human brain.

Three-dimensional visualization of intact tissues is now being achieved by turning tissues transparent. CLARITY is a unique tissue clearing technique, which features the use of detergents to remove lipids from fixed tissues to achieve optical transparency. To preserve tissue integrity, an acrylamide-based hydrogel has been proposed to embed the tissue. In this study, we examined the rationale behind the use of acrylamide in CLARITY, and presented evidence to suggest that the omission of acrylamide-hydrogel embedding in CLARITY does not alter the preservation of tissue morphology and molecular information in fixed tissues. We therefore propose a novel and simplified workflow for formaldehyde-fixed tissue clearing, which will facilitate the laboratory implementation of this technique. Furthermore, we have investigated the basic tissue clearing process in detail and have highlighted some areas for targeted improvement of technologies essential for the emerging subject of three-dimensional histology.

Advancement of laser microscopy and molecular probing techniques in recent years has led to the development of various tissue clearing technologies to allow us to visualise a large piece of tissue in 3D. Using an adaptation of a novel tissue clearing technique, CLARITY, we were able to visualise vascularisation of tumour in a completely new dimension. This image is a colourcoded depth projection (z-stack depth = 618.76 m) of a piece of clarified mouse brain tissue with human glioma immunostained with CD31 antibodies, showing large penetrating branches of blood vessels at the tumour margin

Aims CLARITY is a novel technique which enables three-dimensional visualization of immunostained tissue for the study of circuitry and spatial interactions between cells and molecules in the brain. In this study, we aimed to compare methodological differences in the application of CLARITY between rodent and large human post mortem brain samples. In addition, we aimed to investigate if this technique could be used to visualize Lewy pathology in a post mortem Parkinson's brain. Methods Rodent and human brain samples were clarified and immunostained using the passive version of the CLARITY technique. Samples were then immersed in different refractive index matching media before mounting and visualizing under a confocal microscope. Results We found that tissue clearing speed using passive CLARITY differs according to species (human vs. rodents), brain region and degree of fixation (fresh vs. formalin-fixed tissues). Furthermore, there were advantages to using specific refractive index matching media. We have applied this technique and have successfully visualized Lewy body inclusions in three dimensions within the nucleus basalis of Meynert, and the spatial relationship between monoaminergic fibres and Lewy pathologies among nigrostriatal fibres in the midbrain without the need for physical serial sectioning of brain tissue. Conclusions The effective use of CLARITY on large samples of human tissue opens up many potential avenues for detailed pathological and morphological studies.

Technological advancement has benefited minimal invasive surgery substantially. In terms of enhanced operative vision, three-dimensional (3D) laparoscopy provides binocular depth perception, which was absent in the past with the conventional two-dimensional (2D) lap-aroscopy. There have been a number of studies comparing the use of 2D and 3D laparoscopy over the years and they were reviewed in this article. Essentially, 3D laparoscopy resulted in better performance time and lower error rates with specific tasks in experimental settings, and this was seen in surgeons across different levels of experience. The advantage in terms of operative and clinical outcome was equivocal, with some studies demonstrated faster op-erating time and less blood loss while others showing similar results. Long-term results in this aspect are scarce. Despite these, surgeons appeared to prefer 3D laparoscopy to the con-ventional 2D imaging system. Nevertheless, there are still questions to be answered and the role of 3D laparoscopy can be better defined with evidence from future studies.

Three-dimensional (3D) histology utilizes tissue clearing techniques to turn intact tissues transparent, allowing rapid interrogation of tissue architecture in three dimensions. In this article, we summarized the available tissue clearing methods and classified them according to their physicochemical principles of operation, which provided a framework for one to choose the best techniques for various research settings. Recent attempts in addressing various questions regarding the degenerating and regenerating nervous system have been promising with the use of 3D histological techniques.