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Abstract Tumor heterogeneity and tumor evolution contribute to cancer treatment failure. To understand how selective pressures drive heterogeneous tumor evolution, it would be useful to image multiple important components and tumor subclones in vivo. We propose a supercontinuum-tailoring two-photon microscope (SCT-TPM) and realize simultaneous observation of nine fluorophores with a single light beam, breaking through the ‘color barrier’ of intravital two-photon fluorescence imaging. It achieves excitation multiplexing only by modulating the phase of fiber supercontinuum (SC), allowing to capture rapid events of multiple targets with maintaining precise spatial alignment. We employ SCT-TPM to visualize the spatiotemporal dynamics of heterogeneous tumor evolution under host immune surveillance, particularly the behaviors and interactions of six tumor subclones, immune cells and vascular network, and thus infer the trajectories of tumor progression and clonal competition. SCT-TPM opens up the possibility of tumor lineage tracking and mechanism exploration in living biological systems.

The miRNA plays a key role in the regulation of hormone signaling in insects. The pathways by which miRNAs affect hormone levels are unclear in the honeybee (Apis mellifera), an indispensable pollinator in nature. In this study, ame-miR-5119 was overexpressed and knocked down in larvae by feeding mimics and inhibitors, respectively, and we determined that ame-miR-5119 regulates hormone signaling through the target gene ecdysis triggering hormone (Eth), which affects the larval-pupal transition of workers. The results showed that ame-miR-5119 with a length of 19 nt targets six genes related to the hormone pathway. We focused on Eth and found that ame-miR-5119 and Eth exhibited reverse expression patterns during the transition from larval to pupal stages in workers. Dual luciferase assay confirmed the negative regulatory between ame-miR-5119 and Eth. Overexpression of ame-miR-5119 decreased the mRNA level of Eth, and the Eth receptor (Ethr) expression was not significantly affected, but the expression levels of juvenile hormone (JH) pathway related genes juvenile hormone acid methyltransferase (Jhamt) and Krüppel homolog 1 (Kr-h1) were significantly reduced. In contrast, knockdown of ame-miR-5119 increased the mRNA level of Eth, and the expression of Ethr, Jhamt and Kr-h1 was significantly upregulated. ame-miR-5119 did not affect larval body weight. The number of larvae overexpressing ame-miR-5119 survived in the prepupal stage was lower than that in the control group, and the number of pupations reduced at 11-day-old. The number of larvae that knocked down ame-miR-5119 survived in the prepupal stage was significantly higher than that in the control group, and the number of pupations increased at 11-day-old. These results indicated that ame-miR-5119 negatively regulates the expression of Eth, indirectly inhibits the expression of Ethr, Jhamt, and Kr-h1, and affects the JH biosynthesis, thereby preventing the metamorphic transition from larva to pupa in worker bees. These findings provide evidence that the miRNA regulation of hormone levels in honey bees.

Abstract Amyloid-β (Aβ) readily misfolds into neurotoxic aggregates, generating high levels of reactive oxygen species (ROS), leading to progressive oxidative damage and ultimately cell death. Therefore, simultaneous inhibition of Aβ aggregation and scavenging of ROS may be a promising therapeutic strategy to alleviate Alzheimer’s disease pathology. Based on the previously developed antibody 1F12 that targets all forms of Aβ42, we developed an Aβ42 and ROS dual-targeting nanocomposite using biodegradable mesoporous silica nanoparticles as carriers to load ultra-small cerium oxide nanocrystals (bMSNs@Ce-1F12). By modifying the brain-targeted rabies virus glycoprotein 29 (RVG29-bMSNs@Ce-1F12), this intelligent nanocomposite can efficiently target brain Aβ-rich regions. Combined with peripheral and central nervous system treatments, RVG29-bMSNs@Ce-1F12 can significantly alleviate AD symptoms by inhibiting Aβ42 misfolding, accelerating Aβ42 clearance, and scavenging ROS. Furthermore, this synergistic effect of ROS scavenging and Aβ clearance exhibited by this Aβ42 and ROS dual-targeted strategy also reduced the burden of hyperphosphorylated tau, alleviated glial cell activation, and ultimately improved cognitive function in APP/PS1 mice. Our findings indicate that RVG29-bMSNs@Ce-1F12 is a promising nanodrug that can facilitate multi-target treatment of AD.

Abstract Meningeal lymphatic vessels (mLVs) have been shown to be involved in amyloid beta (Aβ) clearance, which is considered as a potential therapeutic target for Alzheimer’s disease (AD). In this study, based on the superficial spatial distribution of mLVs, a near-infrared light is employed to modulate lymphatic drainage, significantly improving cognition of both aged and AD (5xFAD and APP/PS1) mice, and alleviating AD-associated pathology by reducing Aβ deposition, neuroinflammation and neuronal damage. Furthermore, transmission electron microscopy imaging and RNA sequencing data indicate amelioration of mitochondrial metabolism and cellular junction of meningeal lymphatic endothelial cells (mLECs) by light modulation. These studies collectively suggest that near-infrared light treatment can improve cognitive function by strengthening scavenging ability of mLVs through restoring mLEC function. In conclusion, lymphatic drainage potentiation by light promotes pathological remission and cognitive enhancement in aging and AD mouse models, which offers a potential amelioration strategy for neurodegenerative diseases.

Abstract One intrinsic yet critical issue that troubles the field of fluorescence microscopy ever since its introduction is the unmatched resolution in the lateral and axial directions (i.e., resolution anisotropy), which severely deteriorates the quality, reconstruction, and analysis of 3D volume images. By leveraging the natural anisotropy, we present a deep self-learning method termed Self-Net that significantly improves the resolution of axial images by using the lateral images from the same raw dataset as rational targets. By incorporating unsupervised learning for realistic anisotropic degradation and supervised learning for high-fidelity isotropic recovery, our method can effectively suppress the hallucination with substantially enhanced image quality compared to previously reported methods. In the experiments, we show that Self-Net can reconstruct high-fidelity isotropic 3D images from organelle to tissue levels via raw images from various microscopy platforms, e.g., wide-field, laser-scanning, or super-resolution microscopy. For the first time, Self-Net enables isotropic whole-brain imaging at a voxel resolution of 0.2 × 0.2 × 0.2 μm3, which addresses the last-mile problem of data quality in single-neuron morphology visualization and reconstruction with minimal effort and cost. Overall, Self-Net is a promising approach to overcoming the inherent resolution anisotropy for all classes of 3D fluorescence microscopy.

The sensorimotor cortex participates in diverse functions with different reciprocally connected subregions and projection-defined pyramidal neuron types therein, while the fundamental organizational logic of its circuit elements at the single-cell level is still largely unclear. Here, using mouse Cre driver lines and high-resolution whole-brain imaging to selectively trace the axons and dendrites of cortical pyramidal tract (PT) and intratelencephalic (IT) neurons, we reconstructed the complete morphology of 1,023 pyramidal neurons and generated a projectome of 6 subregions within the sensorimotor cortex. Our morphological data revealed substantial hierarchical and layer differences in the axonal innervation patterns of pyramidal neurons. We found that neurons located in the medial motor cortex had more diverse projection patterns than those in the lateral motor and sensory cortices. The morphological characteristics of IT neurons in layer 5 were more complex than those in layer 2/3. Furthermore, the soma location and morphological characteristics of individual neurons exhibited topographic correspondence. Different subregions and layers were composed of different proportions of projection subtypes that innervate downstream areas differentially. While the axonal terminals of PT neuronal population in each cortical subregion were distributed in specific subdomains of the superior colliculus (SC) and zona incerta (ZI), single neurons selectively innervated a combination of these projection targets. Overall, our data provide a comprehensive list of projection types of pyramidal neurons in the sensorimotor cortex and begin to unveil the organizational principle of these projection types in different subregions and layers.

Abstract Background Dysbiosis or imbalance of gut microbiota in Alzheimer's disease (AD) affects the production of short-chain fatty acids (SCFAs), whereas exogenous SCFAs supplementation exacerbates brain Aβ burden in APP/PS1 mice. Bifidobacterium is the main producer of SCFAs in the gut flora, but oral administration of Bifidobacterium is ineffective due to strong acids and bile salts in the gastrointestinal tract. Therefore, regulating the levels of SCFAs in the gut is of great significance for AD treatment. Methods We investigated the feasibility of intranasal delivery of MSNs-Bifidobacterium (MSNs-Bi) to the gut and their effect on behavior and brain pathology in APP/PS1 mice. Results Mesoporous silica nanospheres (MSNs) were efficiently immobilized on the surface of Bifidobacterium. After intranasal administration, fluorescence imaging of MSNs-Bi in the abdominal cavity and gastrointestinal tract revealed that intranasally delivered MSNs-Bi could be transported through the brain to the peripheral intestine. Intranasal administration of MSNs-Bi not only inhibited intestinal inflammation and reduced brain Aβ burden but also improved olfactory sensitivity in APP/PS1 mice. Conclusions These findings suggested that restoring the balance of the gut microbiome contributes to ameliorating cognitive impairment in AD, and that intranasal administration of MSNs-Bi may be an effective therapeutic strategy for the prevention of AD and intestinal disease.

Abstract Adaptive optics (AO) is a powerful tool for optical microscopy to counteract the effects of optical aberrations and improve the imaging performance in biological tissues. The diversity of sample characteristics entails the use of different AO schemes to measure the underlying aberrations. Here, we present an indirect wavefront sensing method leveraging a virtual imaging scheme and a structural-similarity-based shift measurement algorithm to enable aberration measurement using intrinsic structures even with temporally varying signals. We achieved high-resolution two-photon imaging in a variety of biological samples, including fixed biological tissues and living animals, after aberration correction. We present AO-incorporated subtractive imaging to show that our method can be readily integrated with resolution enhancement techniques to obtain higher resolution in biological tissues. The robustness of our method to signal variation is demonstrated by both simulations and aberration measurement on neurons exhibiting spontaneous activity in a living larval zebrafish.

High-definition fluorescent micro-optical sectioning tomography is employed to resolve fine structures of multiple vessel types in the intact liver.

Automated evaluation of all glomeruli throughout the whole kidney is essential for the comprehensive study of kidney function as well as understanding the mechanisms of kidney disease and development. The emerging large-volume microscopic optical imaging techniques allow for the acquisition of mouse whole-kidney 3D datasets at a high resolution. However, fast and accurate analysis of massive imaging data remains a challenge. Here, we propose a deep learning-based segmentation method called FastCellpose to efficiently segment all glomeruli in whole mouse kidneys. Our framework is based on Cellpose, with comprehensive optimization in network architecture and the mask reconstruction process. By means of visual and quantitative analysis, we demonstrate that FastCellpose can achieve superior segmentation performance compared to other state-of-the-art cellular segmentation methods, and the processing speed was 12-fold higher than before. Based on this high-performance framework, we quantitatively analyzed the development changes of mouse glomeruli from birth to maturity, which is promising in terms of providing new insights for research on kidney development and function.

Organoids can recapitulate human-specific phenotypes and functions in vivo and have great potential for research in development, disease modeling, and drug screening. Due to the inherent variability among organoids, experiments often require a large sample size. Embedding, staining, and imaging each organoid individually require a lot of reagents and time. Hence, there is an urgent need for fast and efficient methods for analyzing the phenotypic changes in organoids in batches. Here, we provide a comprehensive strategy for array embedding, staining, and imaging of cerebral organoids in both agarose sections and in 3D to analyze the spatial distribution of biomarkers in organoids in situ. We constructed several disease models, particularly an aging model, as examples to demonstrate our strategy for the investigation of the phenotypic analysis of organoids. We fabricated an array mold to produce agarose support with microwells, which hold organoids in place for live/dead imaging. We performed staining and imaging of sectioned organoids embedded in agarose and 3D imaging to examine phenotypic changes in organoids using fluorescence micro-optical sectioning tomography (fMOST) and whole-mount immunostaining. Parallel studies of organoids in arrays using the same staining and imaging parameters enabled easy and reliable comparison among different groups. We were able to track all the data points obtained from every organoid in an embedded array. This strategy could help us study the phenotypic changes in organoids in disease models and drug screening.

Short-term memory deficits are associated with prefrontal cortex dysfunction in Alzheimer’s disease. Here, the authors assessed extratelencephalic projection (ET) neurons and found reduced ET neural activity in the medial prefrontal cortex (mPFC) and showed ET neurons received fewer cholinergic inputs from the basal forebrain in 5×FAD mice which led to object recognition memory deficits.

Cells are the basic unit of human organs that are not fully understood. The revolutionary advancements of optical imaging allowed us to observe single cells in whole organs, revealing the complicated composition of cells with spatial information. Therefore, in this review, we revisit the principles of optical contrast related to those biomolecules and the optical techniques that transform optical contrast into detectable optical signals. Then, we describe optical imaging to achieve three-dimensional spatial discrimination for biological tissues. Due to the milky appearance of tissues, the spatial information blurred deep in the whole organ. Fortunately, strategies developed in the last decade could circumvent this issue and lead us into a new era of investigation of the cells with their original spatial information.

BackgroundThe APLAID syndrome is a rare primary immunodeficiency caused by gain-of-function mutations in the PLCG2 gene. We present a 7-year-old APLAID patient who has recurrent blistering skin lesions, skin infections in the perineum, a rectal perineal fistula, and inflammatory bowel disease.MethodsTo determine the genetic cause of our patient, WES and bioinformatics analysis were performed. Flow cytometry was used for phenotyping immune cell populations in peripheral blood. Cytokines released into plasma were analyzed using protein chip technology. The PBMCs of patient and a healthy child were subjected to single-cell RNA-sequencing analysis.ResultsThe patient carried a novel de novo missense mutation c.2534T>C in exon 24 of the PLCG2 gene that causes a leucine to serine amino acid substitution (p.Leu845Ser). Bioinformatics analysis revealed that this mutation had a negative impact on the structure of the PLCγ2 protein, which is highly conserved in many other species. Immunophenotyping by flow cytometry revealed that in addition to the typical decrease in circulating memory B cells, the levels of myeloid dendritic cells (mDCs) in the children’s peripheral blood were significantly lower, as were the CD4+ effector T cells induced by their activation. Single-cell sequencing revealed that the proportion of different types of cells in the peripheral blood of the APLAID patient changed.ConclusionsWe present the first case of APLAID with severely reduced myeloid dendritic cells carrying a novel PLCG2 mutation, and conducted a comprehensive analysis of immunological features in the ALPAID patient, which has not been mentioned in previous reports. This study expands the spectrum of APLAID-associated immunophenotype and genotype. The detailed immune analyses in this patient may provide a basis for the development of targeted therapies for this severe autoinflammatory disease.

Cao et al. present a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA), used to develop AmpFISH workflows. Their method is suitable for labelling to both individual mRNA molecules and chromosomes and can also be multiplexed with immunofluorescence, allowing for sensitive RNA/DNA detection.

Abstract Axonal projection conveys neural information. The divergent and diverse projections of individual neurons imply the complexity of information flow. It is necessary to investigate the relationship between the projection and functional information at the single neuron level for understanding the rules of neural circuit assembly, but a gap remains due to a lack of methods to map the function to whole‐brain projection. Here an approach is developed to bridge two‐photon calcium imaging in vivo with high‐resolution whole‐brain imaging based on sparse labeling with the genetically encoded calcium indicator GCaMP6. Reliable whole‐brain projections are captured by the high‐definition fluorescent micro‐optical sectioning tomography (HD‐fMOST). A cross‐modality cell matching is performed and the functional annotation of whole‐brain projection at the single‐neuron level (FAWPS) is obtained. Applying it to the layer 2/3 (L2/3) neurons in mouse visual cortex, the relationship is investigated between functional preferences and axonal projection features. The functional preference of projection motifs and the correlation between axonal length in MOs and neuronal orientation selectivity, suggest that projection motif‐defined neurons form a functionally specific information flow, and the projection strength in specific targets relates to the information clarity. This pipeline provides a new way to understand the principle of neuronal information transmission.

Obtaining various structures of the entire mature heart at single-cell resolution is highly desired in cardiac studies; however, effective methodologies are still lacking. Here, we propose a pipeline for labeling and imaging myocardial and vascular structures. In this pipeline, the myocardium is counterstained using fluorescent dyes and the cardiovasculature is labeled using transgenic markers. High-definition dual-color fluorescence micro-optical sectioning tomography is used to perform heart-wide tissue imaging, enabling the acquisition of whole-heart data at a voxel resolution of 0.32 × 0.32 × 1 μm3. Obtained structural data demonstrated the superiority of the pipeline. In particular, the three-dimensional morphology and spatial arrangement of reconstructed cardiomyocytes were revealed, and high-resolution vascular data helped determine differences in the features of endothelial cells and complex coiled capillaries. Our pipeline can be used in cardiac studies for examining the structures of the entire heart at the single-cell level.

Summary: Optical visualization of complex microstructures in the entire organ is essential for biomedical research. However, the existing methods fail to accurately acquire the detailed microstructures of whole organs with good morphological and biochemical preservation. This study proposes a cryo-fluorescence micro-optical sectioning tomography (cryo-fMOST) to image whole organs in three dimensions (3D) with submicron resolution. The system comprises a line-illumination microscope module, cryo-microtome, three-stage refrigeration module, and heat insulation device. To demonstrate the imaging capacity and wide applicability of the system, we imaged and reconstructed various organs of mice in 3D, including the healthy tongue, kidney, and brain, as well as the infarcted heart. More importantly, imaged brain slices were performed sugar phosphates determination and fluorescence in situ hybridization imaging to verify the compatibility of multi-omics measurements. The results demonstrated that cryo-fMOST is capable of acquiring high-resolution morphological details of various whole organs and may be potentially useful for spatial multi-omics.

Abstract Background Neurons are the basic structural unit of the brain, and their morphology is a key determinant of their classification. The morphology of a neuronal circuit is a fundamental component in neuron modeling. Recently, single-neuron morphologies of the whole brain have been used in many studies. The correctness and completeness of semimanually traced neuronal morphology are credible. However, there are some inaccuracies in semimanual tracing results. The distance between consecutive nodes marked by humans is very long, spanning multiple voxels. On the other hand, the nodes are marked around the centerline of the neuronal fiber, not on the centerline. Although these inaccuracies do not seriously affect the projection patterns that these studies focus on, they reduce the accuracy of the traced neuronal skeletons. These small inaccuracies will introduce deviations into subsequent studies that are based on neuronal morphology files. Results We propose a neuronal digital skeleton optimization method to evaluate and make fine adjustments to a digital skeleton after neuron tracing. Provided that the neuronal fiber shape is smooth and continuous, we describe its physical properties according to two shape restrictions. One restriction is designed based on the grayscale image, and the other is designed based on geometry. These two restrictions are designed to finely adjust the digital skeleton points to the neuronal fiber centerline. With this method, we design the three-dimensional shape restriction workflow of neuronal skeleton adjustment computation. The performance of the proposed method has been quantitatively evaluated using synthetic and real neuronal image data. The results show that our method can reduce the difference between the traced neuronal skeleton and the centerline of the neuronal fiber. Furthermore, morphology metrics such as the neuronal fiber length and radius become more precise. Conclusions This method can improve the accuracy of a neuronal digital skeleton based on traced results. The greater the accuracy of the digital skeletons that are acquired, the more precise the neuronal morphologies that are analyzed will be.

The bee venom melittin has anti-tumor properties but is unsuitable for therapeutic use on its own. Here, the authors generate melittin-nanoparticles and demonstrate that the nanoparticles reduce tumor growth and generate an anti-tumor immune response.

The glutamatergic and GABAergic neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) mediated diverse brain functions. However, their whole-brain neural connectivity has not been comprehensively mapped. Here we used the virus tracers to characterize the whole-brain inputs and outputs of glutamatergic and GABAergic neurons in VTA and SNc. We found that these neurons received similar inputs from upstream brain regions, but some quantitative differences were also observed. Neocortex and dorsal striatum provided a greater share of input to VTA glutamatergic neurons. Periaqueductal gray and lateral hypothalamic area preferentially innervated VTA GABAergic neurons. Specifically, superior colliculus provided the largest input to SNc glutamatergic neurons. Compared to input patterns, the output patterns of glutamatergic and GABAergic neurons in the VTA and SNc showed significant preference to different brain regions. Our results laid the anatomical foundation for understanding the functions of cell-type-specific neurons in VTA and SNc.

The dorsal raphe nucleus (DR) and median raphe nucleus (MR) contain populations of glutamatergic and GABAergic neurons that regulate diverse behavioral functions. However, their whole-brain input-output circuits remain incompletely elucidated. We used viral tracing combined with fluorescence micro-optical sectioning tomography to generate a comprehensive whole-brain atlas of inputs and outputs of glutamatergic and GABAergic neurons in the DR and MR. We found that these neurons received inputs from similar upstream brain regions. The glutamatergic and GABAergic neurons in the same raphe nucleus had divergent projection patterns with differences in critical brain regions. Specifically, MR glutamatergic neurons projected to the lateral habenula through multiple pathways. Correlation and cluster analysis revealed that glutamatergic and GABAergic neurons in the same raphe nucleus received heterogeneous inputs and sent different collateral projections. This connectivity atlas further elucidates the anatomical architecture of the raphe nuclei, which could facilitate better understanding of their behavioral functions.

Although amyloid-β42 (Aβ42) has been used as one of the core biomarkers for Alzheimer’s disease (AD) diagnosis, the dynamic changes of its different forms in the brain, blood, and even intestines and its correlation with the progression of AD disease remain obscure. Herein, we screened Aβ42-specific preferred antibody pairs 1F12/1F12 and 1F12/2C6 to accurately detect Aβ42 types using sandwich ELISA, including total Aβ42, Aβ42 oligomers (Aβ42Os), and Aβ42 monomers (Aβ42Ms). The levels of Aβ42 species in the brain, blood, and intestines of different aged APP/PS1 mice were quantified to study their correlation with AD progression. Total Aβ42 levels in the blood were not correlated with AD progression, but Aβ42Ms level in the blood of 9-month-old APP/PS1 mice was significantly reduced, and Aβ42Os level in the brain was significantly elevated compared to 3-month-old APP/PS1, demonstrating that the levels of Aβ42Ms and Aβ42Os in the blood and brain were correlated with AD progression. Interestingly, in 9-month-old APP/PS1 mice, the level of Aβ42 in the intestine was higher than that in 3-month-old APP/PS1 mice, indicating that the increased level of Aβ42 in the gastrointestinal organs may also be related to the progression of AD. Meanwhile, changes in the gut microbiota composition of APP/PS1 mice with age were also observed. Therefore, the increase in Aβ derived from intestinal tissues and changes in microbiome composition can be used as a potential early diagnosis tool for AD, and further used as an indicator of drug intervention to reduce brain amyloid.

Reconstructing the full shape of neurons is a major informatics challenge as it requires handling huge whole-brain imaging datasets. Here the authors present an open-source virtual reality annotation system for precise and efficient data production of neuronal shapes reconstructed from whole brains.

Liver sinusoidal endothelial cells are known to promote immune tolerance in liver. Here, the authors target these cells using melittin nanoparticles and show alterations in the liver immune environment and suppression of liver metastases.

Abstract Background Microglia/macrophages (M/Ms) with multiple functions derived from distinct activation states are key surveillants maintaining brain homeostasis. However, their activation status and role during the brain metastasis of malignant tumors have been poorly characterized. Methods Heterozygous CX3CR1-GFP transgenic mice were used to visualize the dynamic changes of M/Ms during the development of experimental brain metastasis through long-term intravital imaging equipped with redesigned bilateral cranial windows. The occurrence of experimental brain metastasis was evaluated after M/Ms were depleted with PLX3397, a CSF-1R inhibitor. The possible mediators of M/Ms in facilitating the brain metastasis were determined using reverse transcription-PCR, immunofluorescence, correlational analysis, and MMP inhibition. Results Here, we showed that M/Ms were persistently activated and facilitated the formation of melanoma brain metastasis in vivo. We observed that M/Ms gradually and massively accumulated in the metastasis, with a 2.89-fold increase. To precisely depict the dynamic changes in the activation state of M/Ms, we defined the branching parameter to quantify their morphological alterations. The quantitative data showed that the extent of activation of M/Ms in metastatic foci was enhanced, with a 2.27-fold increase from day 1 to day 21. Along with the activation, the M/Ms increased their moving velocity (4.15-fold) and established a rapid, confined, and discontinuous motility behavior. The occurrence of melanoma brain metastasis was significantly hindered under M/M elimination, indicating the key role of M/Ms in the experimental brain metastasis. Interestingly, we found that M/Ms highly expressed matrix metalloproteinase 3 (MMP3), which were strongly correlated with M/M activation and the decrease of tight junction protein zonula occludens-1 (ZO-1). An MMP inhibitor moderately decreased the occurrence of melanoma brain metastasis, suggesting that MMP3 secreted by M/Ms may facilitate melanoma cell growth. Conclusions Our results indicated that the activated M/Ms were essential in the development of melanoma brain metastasis, suggesting that M/Ms are a potential therapeutic target for tumor brain metastasis.

Cerebral organoids recapitulate in vivo phenotypes and physiological functions of the brain and have great potential in studying brain development, modeling diseases, and conducting neural network research. It is essential to obtain whole-mount three-dimensional (3D) images of cerebral organoids at cellular levels to explore their characteristics and applications. Existing histological strategies sacrifice inherent spatial characteristics of organoids, and the strategy for volume imaging and 3D analysis of entire organoids is urgently needed. Here, we proposed a high-resolution imaging pipeline based on fluorescent labeling by viral transduction and 3D immunostaining with fluorescence micro-optical sectioning tomography (fMOST). We were able to image intact organoids using our pipeline, revealing cytoarchitecture information of organoids and the spatial localization of neurons and glial fibrillary acidic protein positive cells (GFAP+ cells). We performed single-cell reconstruction to analyze the morphology of neurons and GFAP+ cells. Localization and quantitative analysis of cortical layer markers revealed heterogeneity of organoids. This pipeline enabled acquisition of high-resolution spatial information of millimeter-scale organoids for analyzing their cell composition and morphology.

Abstract Tissue optical clearing techniques have provided important tools for large‐volume imaging. Aqueous‐based clearing methods are known for good fluorescence preservation and scalable size maintenance, but are limited by long incubation time, insufficient clearing performance, or requirements for specialized devices. Additionally, few clearing methods are compatible with widely used lipophilic dyes while maintaining high clearing performance. Here, to address these issues, m‐xylylenediamine (MXDA) is firstly introduced into tissue clearing and used to develop a rapid, highly efficient aqueous clearing method with robust lipophilic dyes compatibility, termed MXDA‐based Aqueous Clearing System (MACS). MACS can render whole adult brains highly transparent within 2.5 days and is also applicable for other intact organs. Meanwhile, MACS possesses ideal compatibility with multiple probes, especially for lipophilic dyes. MACS achieves 3D imaging of the intact neural structures labeled by various techniques. Combining MACS with DiI labeling, MACS allows reconstruction of the detailed vascular structures of various organs and generates 3D pathology of glomeruli tufts in healthy and diabetic kidneys. Therefore, MACS provides a useful method for 3D mapping of intact tissues and is expected to facilitate morphological, physiological, and pathological studies of various organs.

Mapping the cytoarchitecture of the whole brain can reveal the organizational logic of neural systems. However, this remains a significant challenge, especially for gyrencephalic brains with a large volume. Here we propose an integrated pipeline for generating a cytoarchitectonic atlas with single-cell resolution of the whole brain. To analyze a large-volume brain, we used a modified en-bloc Nissl staining protocol to achieve uniform staining of large-scale brain specimens from ferret (Mustela putorius furo). By combining whole-brain imaging and big data processing, we established strategies for parsing cytoarchitectural information at a voxel resolution of 0.33 μm × 0.33 μm × 1 μm and terabyte-scale data analysis. Using the cytoarchitectonic datasets for adult ferret brain, we identified giant pyramidal neurons in ferret brains and provide the first report of their morphological diversity, neurochemical phenotype, and distribution patterns in the whole brain in three dimensions. This pipeline will facilitate studies on the organization and development of the mammalian brains, from that of rodents to the gyrencephalic brains of ferret and even primates.

Summary: Ventral hippocampus (vHIP) and medial prefrontal cortex (mPFC) are both critical regions for social behaviors. However, how their interactions affect social behavior is not well understood. By viral tracing, optogenetics, chemogenetics, and fiber photometry, we demonstrated that inhibition of vHIP or direct projections from vHIP to mPFC impaired social memory expression. Via rabies retrograde tracing, we found that all three major GABAergic neurons in mPFC received direct inputs from vHIP. Activation of parvalbumin positive (PV+) neurons in mPFC but not somatostatin positive (SST+) neurons can rescue the social memory impairment caused by vHIP inhibition. Furthermore, fiber photometry results demonstrated that social behaviors preferentially recruited PV+ neurons and inhibition of hippocampal neurons disrupted the activity of PV+ neurons during social interactions. These results revealed a new mechanism of how vHIP and mPFC regulate social behavior in complementarity with the existing neural circuitry mechanism. : Behavioral Neuroscience; Neuroscience; Systems Neuroscience Subject Areas: Behavioral Neuroscience, Neuroscience, Systems Neuroscience

The segmentation of brain region contours in three dimensions is critical for the analysis of different brain structures, and advanced approaches are emerging continuously within the field of neurosciences. With the development of high-resolution micro-optical imaging, whole-brain images can be acquired at the cellular level. However, brain regions in microscopic images are aggregated by discrete neurons with blurry boundaries, the complex and variable features of brain regions make it challenging to accurately segment brain regions. Manual segmentation is a reliable method, but is unrealistic to apply on a large scale. Here, we propose an automated brain region segmentation framework, DeepBrainSeg, which is inspired by the principle of manual segmentation. DeepBrainSeg incorporates three feature levels to learn local and contextual features in different receptive fields through a dual-pathway convolutional neural network (CNN), and to provide global features of localization by image registration and domain-condition constraints. Validated on biological datasets, DeepBrainSeg can not only effectively segment brain-wide regions with high accuracy (Dice ratio > 0.9), but can also be applied to various types of datasets and to datasets with noises. It has the potential to automatically locate information in the brain space on the large scale.

Serotonin neurons of the dorsal and median raphe nuclei (DR, MR) collectively innervate the entire forebrain and midbrain, modulating diverse physiology and behavior. To gain a fundamental understanding of their molecular heterogeneity, we used plate-based single-cell RNA-sequencing to generate a comprehensive dataset comprising eleven transcriptomically distinct serotonin neuron clusters. Systematic in situ hybridization mapped specific clusters to the principal DR, caudal DR, or MR. These transcriptomic clusters differentially express a rich repertoire of neuropeptides, receptors, ion channels, and transcription factors. We generated novel intersectional viral-genetic tools to access specific subpopulations. Whole-brain axonal projection mapping revealed that DR serotonin neurons co-expressing vesicular glutamate transporter-3 preferentially innervate the cortex, whereas those co-expressing thyrotropin-releasing hormone innervate subcortical regions in particular the hypothalamus. Reconstruction of 50 individual DR serotonin neurons revealed diverse and segregated axonal projection patterns at the single-cell level. Together, these results provide a molecular foundation of the heterogenous serotonin neuronal phenotypes.

The excitatory neurons in the visual cortex are of great significance for us in understanding brain functions. However, the diverse neuron types and their morphological properties have not been fully deciphered. In this paper, we applied the brain-wide positioning system (BPS) to image the entire brain of two Thy1-eYFP H-line male mice at 0.2 μm × 0.2 μm × 1 μm voxel resolution. A total of 103 neurons were reconstructed in layers 5 and 6 of the visual cortex with single-axon-level resolution. Based on the complete topology of neurons and the inherent positioning function of the imaging method, we classified the observed neurons into six types according to their apical dendrites and somata location: star pyramidal cells in layer 5 (L5-sp), slender-tufted pyramidal cells in layer 5 (L5-st), tufted pyramidal cells in layer 5 (L5-tt), spiny stellate-like cells in layer 6 (L6-ss), star pyramidal cells in layer 6 (L6-sp), and slender-tufted pyramidal cells in layer 6 (L6-st). By examining the axonal projection patterns of individual neurons, they can be categorized into three modes: ipsilateral circuit connection neurons, callosal projection neurons and corticofugal projection neurons. Correlating the two types of classifications, we have found that there are at least two projection modes comprised in the former defined neuron types except for L5-tt. On the other hand, each projection mode may consist of four dendritic types defined in this study. The axon projection mode only partially correlates with the apical dendrite feature. This work has demonstrated a paradigm for resolving the visual cortex through single-neuron-level quantification and has shown potential to be extended to reveal the connectome of other defined sensory and motor systems.

Fine morphological reconstruction of individual neurons across the entire brain is essential for mapping brain circuits. Inference of presynaptic axonal boutons, as a key part of single-neuron fine reconstruction, is critical for interpreting the patterns of neural circuit wiring schemes. However, automated bouton identification remains challenging for current neuron reconstruction tools, as they focus mainly on neurite skeleton drawing and have difficulties accurately quantifying bouton morphology. Here, we developed an automated method for recognizing single-neuron axonal boutons in whole-brain fluorescence microscopy datasets. The method is based on deep convolutional neural networks and density-peak clustering. High-dimensional feature representations of bouton morphology can be learned adaptively through convolutional networks and used for bouton recognition and subtype classification. We demonstrate that the approach is effective for detecting single-neuron boutons at the brain-wide scale for both long-range pyramidal projection neurons and local interneurons.

Summary: Parsing diverse nerve cells into biological types is necessary for understanding neural circuit organization. Morphology is an intuitive criterion for neuronal classification and a proxy of connectivity, but morphological diversity and variability often preclude resolving the granularity of neuron types. Combining genetic labeling with high-resolution, large-volume light microscopy, we established a single neuron anatomy platform that resolves, registers, and quantifies complete neuron morphologies in the mouse brain. We discovered that cortical axo-axonic cells (AACs), a cardinal GABAergic interneuron type that controls pyramidal neuron (PyN) spiking at axon initial segments, consist of multiple subtypes distinguished by highly laminar-specific soma position and dendritic and axonal arborization patterns. Whereas the laminar arrangements of AAC dendrites reflect differential recruitment by input streams, the laminar distribution and local geometry of AAC axons enable differential innervation of PyN ensembles. This platform will facilitate genetically targeted, high-resolution, and scalable single neuron anatomy in the mouse brain. : Wang et al. combine mouse genetic labeling with high-resolution, large-volume light microscopy and establish a single-neuron anatomy platform. They show that cortical axo-axonic cells, a GABAergic interneuron type that innervates pyramidal neurons at axon initial segments, consist of multiple subtypes distinguished by laminar position and dendritic and axonal arborization. Keywords: GABAergic interneurons, axo-axonic cells, AACs, chandelier cells, ChCs, neuron subtypes, genetic single neuron anatomy, gSNA, fMOST

Reconstruction of neuronal morphology from images involves mainly the extraction of neuronal skeleton points. It is an indispensable step in the quantitative analysis of neurons. Due to the complex morphology of neurons, many widely used tracing methods have difficulties in accurately acquiring skeleton points near branch points or in structures with tortuosity. Here, we propose two models to solve these problems. One is based on an L1-norm minimization model, which can better identify tortuous structure, namely, a local structure with large curvature skeleton points; the other detects an optimized branch point by considering the combination patterns of all neurites that link to this point. We combined these two models to achieve optimized skeleton detection for a neuron. We validate our models in various datasets including MOST and BigNeuron. In addition, we demonstrate that our method can optimize the traced skeletons from large-scale images. These characteristics of our approach indicate that it can reduce manual editing of traced skeletons and help to accelerate the accurate reconstruction of neuronal morphology.

High-throughput imaging methods for brain-wide connectome mapping with precise location reference have been lacking. Here authors report a method that allows simultaneous acquisition of fluorescently labelled neurons and cytoarchitectural landmarks in the same mouse brain at the single-cell resolution.

Understanding amazingly complex brain functions and pathologies requires a complete cerebral vascular atlas in stereotaxic coordinates. Making a precise atlas for cerebral arteries and veins has been a century-old objective in neuroscience and neuropathology. Using micro-optical sectioning tomography (MOST) with a modified Nissl staining method, we acquired five mouse brain data sets containing arteries, veins, and microvessels. Based on the brain-wide vascular spatial structures and brain regions indicated by cytoarchitecture in one and the same mouse brain, we reconstructed and annotated the vascular system atlas of both arteries and veins of the whole mouse brain for the first time. The distributing patterns of the vascular system within the brain regions were acquired and our results show that the patterns of individual vessels are different from each other. Reconstruction and statistical analysis of the microvascular network, including derivation of quantitative vascular densities, indicate significant differences mainly in vessels with diameters less than 8 μm and large than 20 μm across different brain regions. Our precise cerebral vascular atlas provides an important resource and approach for quantitative studies of brain functions and diseases.

Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis.

Three-dimensional imaging of whole mammalian brains at single-neuron resolution has generated terabyte (TB)- and even petabyte (PB)-sized datasets. Due to their size, processing these massive image datasets can be hindered by the computer hardware and software typically found in biological laboratories. To fill this gap, we have developed an efficient platform named TDat, which adopts a novel data reformatting strategy by reading cuboid data and employing parallel computing. In data reformatting, TDat is more efficient than any other software. In data accessing, we adopted parallelization to fully explore the capability for data transmission in computers. We applied TDat in large-volume data rigid registration and neuron tracing in whole-brain data with single-neuron resolution, which has never been demonstrated in other studies. We also showed its compatibility with various computing platforms, image processing software and imaging systems.

Corticotropin-releasing hormone (CRH), with widespread expression in the brain, plays a key role in modulating a series of behaviors, including anxiety, arousal, motor function, learning and memory. Previous studies have focused on some brain regions with densely distributed CRH neurons such as paraventricular hypothalamic nucleus (PVH) and bed nuclei of the stria terminalis (BST) and revealed some basic structural and functional knowledge of CRH neurons. However, there is no systematic analysis of brain-wide distribution of CRH neurons. Here, we performed a comprehensive study of CRH neurons in CRH-IRES-Cre;Ai3 mice via automatic imaging and stereoscopic cell counting in a whole mouse brain. We acquired four datasets of the CRH distributions with co-localized cytoarchitecture at a voxel resolution of 0.32 μm × 0.32 μm × 2 μm using brain-wide positioning system (BPS). Next, we precisely located and counted the EYFP-labeled neurons in different regions according to propidium iodide counterstained anatomical reference using Neuronal Global Position System. In particular, dense EYFP expression was found in piriform area, BST, central amygdalar nucleus, PVH, Barrington’s nucleus, and inferior olivary complex. Considerable CRH neurons were also found in main olfactory bulb, medial preoptic nucleus, pontine gray, tegmental reticular nucleus, external cuneate nucleus, and midline thalamus. We reconstructed and compared the soma morphology of CRH neurons in 11 brain regions. The results demonstrated that CRH neurons had regional diversities of both cell distribution and soma morphology. This anatomical knowledge enhances the current understanding of the functions of CRH neurons. These results also demonstrated the ability of our platform to accurately orient, reconstruct and count neuronal somas in three-dimension for type-specific neurons in the whole brain, making it feasible to answer the fundamental neuroscience question of exact numbers of various neurons in the whole brain.

With the development of hybrid imaging scanners, micro-CT is widely used in locating abnormalities, studying drug metabolism, and providing structural priors to aid image reconstruction in functional imaging. Due to the low contrast of soft tissues, segmentation of soft tissue organs from mouse micro-CT images is a challenging problem. In this paper, we propose a mouse segmentation scheme based on dynamic contrast enhanced micro-CT images. With a homemade fast scanning micro-CT scanner, dynamic contrast enhanced images were acquired before and after injection of non-ionic iodinated contrast agents (iohexol). Then the feature vector of each voxel was extracted from the signal intensities at different time points. Based on these features, the heart, liver, spleen, lung, and kidney could be classified into different categories and extracted from separate categories by morphological processing. The bone structure was segmented using a thresholding method. Our method was validated on seven BALB/c mice using two different classifiers: a support vector machine classifier with a radial basis function kernel and a random forest classifier. The results were compared to manual segmentation, and the performance was assessed using the Dice similarity coefficient, false positive ratio, and false negative ratio. The results showed high accuracy with the Dice similarity coefficient ranging from 0.709 ± 0.078 for the spleen to 0.929 ± 0.006 for the kidney.

When neuroscience’s focus moves from molecular and cellular level to systems level, information technology mixes in and cultivates a new branch neuroinformatics. Especially under the investments of brain initiatives all around the world, brain atlases and connectomics are identified as the substructure to understand the brain. We think it is time to call for a potential interdisciplinary subject, brainsmatics, referring to brain-wide spatial informatics science and emphasizing on precise positioning information affiliated to brain-wide connectome, genome, proteome, transcriptome, metabolome, etc. Brainsmatics methodology includes tracing, surveying, visualizing, and analyzing brain-wide spatial information. Among all imaging techniques, optical imaging is the most appropriate solution to achieve whole-brain connectome in consistent single-neuron resolution. This review aims to introduce contributions of optical imaging to brainsmatics studies, especially the major strategies applied in tracing and surveying processes. After discussions on the state-of-the-art technology, the development objectives of optical imaging in brainsmatics field are suggested. We call for a global contribution to the brainsmatics field from all related communities such as neuroscientists, biologists, engineers, programmers, chemists, mathematicians, physicists, clinicians, pharmacists, etc. As the leading approach, optical imaging will, in turn, benefit from the prosperous development of brainsmatics.

The combined-immunotherapy of adoptive cell therapy (ACT) and cyclophosphamide (CTX) is one of the most efficient treatments for melanoma patients. However, no synergistic effects of CTX and ACT on the spatio-temporal dynamics of immunocytes in vivo have been described. Here, we visualized key cell events in immunotherapy-elicited immunoreactions in a multicolor-coded tumor microenvironment, and then established an optimal strategy of metronomic combined-immunotherapy to enhance anti-tumor efficacy. Intravital imaging data indicated that regulatory T cells formed an 'immunosuppressive ring' around a solid tumor. The CTX-ACT combined-treatment elicited synergistic immunoreactions in tumor areas, which included relieving the immune suppression, triggering the transient activation of endogenous tumor-infiltrating immunocytes, increasing the accumulation of adoptive cytotoxic T lymphocytes, and accelerating the infiltration of dendritic cells. These insights into the spatio-temporal dynamics of immunocytes are beneficial for optimizing immunotherapy and provide new approaches for elucidating the mechanisms underlying the involvement of immunocytes in cancer immunotherapy.

Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both ends of the vector and insert. This method can therefore be used as a ligation-independent cloning strategy to generate recombinant constructs rapidly. In addition, we designed and constructed a simple and specific vector from an Escherichia coli plasmid back-bone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method.

Hypoxia is closely related to many diseases and often leads to death. Early detection and identification of the hypoxia causes may help to promptly determine the right rescue plan and reduce the mortality. We proposed a new multiparametric monitoring method employing mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence, regional reflectance, regional cerebral blood flow (CBF), electrocardiography (ECG), and respiration under six kinds of acute hypoxia in four categories to investigate a correlation between the parameter variances and the hypoxia causes. The variation patterns of the parameters were discussed, and the combination of NADH and CBF may contribute to the identification of the causes of hypoxia.