Fluorescence Micro-optical Sectioning Tomography Research Articles

The cytoarchitectonic landscape revealed by deep learning method facilitated precise positioning in mouse neocortex.

Neocortex is a complex structure with different cortical sublayers and regions. However, the precise positioning of cortical regions can be challenging due to the absence of distinct landmarks without special preparation. To address this challenge, we developed a cytoarchitectonic landmark identification pipeline. The fluorescence micro-optical sectioning tomography method was employed to image the whole mouse brain stained by general fluorescent nucleotide dye. A fast 3D convolution network was subsequently utilized to segment neuronal somas in entire neocortex. By approach, the cortical cytoarchitectonic profile and the neuronal morphology were analyzed in 3D, eliminating the influence of section angle. And the distribution maps were generated that visualized the number of neurons across diverse morphological types, revealing the cytoarchitectonic landscape which characterizes the landmarks of cortical regions, especially the typical signal pattern of barrel cortex. Furthermore, the cortical regions of various ages were aligned using the generated cytoarchitectonic landmarks suggesting the structural changes of barrel cortex during the aging process. Moreover, we observed the spatiotemporally gradient distributions of spindly neurons, concentrated in the deep layer of primary visual area, with their proportion decreased over time. These findings could improve structural understanding of neocortex, paving the way for further exploration with this method.

Link Brain-Wide Projectome to Neuronal Dynamics in the Mouse Brain.

Knowledge about the neuronal dynamics and the projectome are both essential for understanding how the neuronal network functions in concert. However, it remains challenging to obtain the neural activity and the brain-wide projectome for the same neurons, especially for neurons in subcortical brain regions. Here, by combining in vivo microscopy and high-definition fluorescence micro-optical sectioning tomography, we have developed strategies for mapping the brain-wide projectome of functionally relevant neurons in the somatosensory cortex, the dorsal hippocampus, and the substantia nigra pars compacta. More importantly, we also developed a strategy to achieve acquiring the neural dynamic and brain-wide projectome of the molecularly defined neuronal subtype. The strategies developed in this study solved the essential problem of linking brain-wide projectome to neuronal dynamics for neurons in subcortical structures and provided valuable approaches for understanding how the brain is functionally organized via intricate connectivity patterns.

Subregion preference in the long-range connectome of pyramidal neurons in the medial prefrontal cortex

BackgroundThe medial prefrontal cortex (mPFC) is involved in complex functions containing multiple types of neurons in distinct subregions with preferential roles. The pyramidal neurons had wide-range projections to cortical and subcortical regions with subregional preferences. Using a combination of viral tracing and fluorescence micro-optical sectioning tomography (fMOST) in transgenic mice, we systematically dissected the whole-brain connectomes of intratelencephalic (IT) and pyramidal tract (PT) neurons in four mPFC subregions.ResultsIT and PT neurons of the same subregion projected to different target areas while receiving inputs from similar upstream regions with quantitative differences. IT and PT neurons all project to the amygdala and basal forebrain, but their axons target different subregions. Compared to subregions in the prelimbic area (PL) which have more connections with sensorimotor-related regions, the infralimbic area (ILA) has stronger connections with limbic regions. The connection pattern of the mPFC subregions along the anterior–posterior axis showed a corresponding topological pattern with the isocortex and amygdala but an opposite orientation correspondence with the thalamus.ConclusionsBy using transgenic mice and fMOST imaging, we obtained the subregional preference whole-brain connectomes of IT and pyramidal tract PT neurons in the mPFC four subregions. These results provide a comprehensive resource for directing research into the complex functions of the mPFC by offering anatomical dissections of the different subregions.

Single-neuron analysis of axon arbors reveals distinct presynaptic organizations between feedforward and feedback projections

The morphology and spatial distribution of axon arbors and boutons are crucial for neuron presynaptic functions. However, the principles governing their whole-brain organization at the single-neuron level remain unclear. We developed a machine-learning method to separate axon arbors from passing axons in single-neuron reconstruction from fluorescence micro-optical sectioning tomography imaging data and obtained 62,374 axon arbors that displayed distinct morphology, spatial patterns, and scaling laws dependent on neuron types and targeted brain areas. Focusing on the axon arbors in the thalamus and cortex, we revealed the segregated spatial distributions and distinct morphology but shared topographic gradients between feedforward and feedback projections. Furthermore, we uncovered an association between arbor complexity and microglia density. Finally, we found that the boutons on terminal arbors show branch-specific clustering with a log-normal distribution that again differed between feedforward and feedback terminal arbors. Together, our study revealed distinct presynaptic structural organizations underlying diverse functional innervation of single projection neurons.

Cerebral Organoid Arrays for Batch Phenotypic Analysis in Sections and Three Dimensions.

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.

Cross-scale tracing of nanoparticles and tumors at the single-cell level using the whole-lung atlas.

Currently, the effectiveness of oncotherapy is limited by tumor heterogeneities, which presents a huge challenge for the development of nanotargeted drug delivery systems (DDSs). Therefore, it is important to resolve the spatiotemporal interactions between tumors and nanoparticles. However, targeting evaluation has been limited by particle visualization due to the gap between whole-organ scale and subcellular precision. Here, a high-precision three-dimensional (3D) visualization of tumor structure based on the micro-optical sectioning tomography (MOST) system and fluorescence MOST (fMOST) system is presented to clarify 3D spatial distribution of nanoparticles within the tumor. We demonstrate that through the MOST/fMOST system, it is possible to reveal multidimensional and cross-scale correlations between the tumor structure and nanoparticle distribution to remodel the tumor microenvironment and explore the structural parameters of vasculature. This visualization methodology provides an accurate assessment of the efficacy, distribution, and targeting efficiency of DDSs for oncotherapy compared to available approaches.

Application of fluorescence micro-optical sectioning tomography in the cerebrovasculature and applicable vascular labeling methods.

Fluorescence micro-optical sectioning tomography (fMOST) is a three-dimensional (3d) imaging method at the mesoscopic level. The whole-brain of mice can be imaged at a high resolution of 0.32 × 0.32 × 1.00 μm3. It is useful for revealing the fine morphology of intact organ tissue, even for positioning the single vessel connected with a complicated vascular network across different brain regions in the whole mouse brain. Featuring its 3d visualization of whole-brain cross-scale connections, fMOST has a vast potential to decipher brain function and diseases. This article begins with the background of fMOST technology including a widespread 3D imaging methods comparison and the basic technical principal illustration, followed by the application of fMOST in cerebrovascular research and relevant vascular labeling techniques applicable to different scenarios.

3D Visualization of Whole Brain Vessels and Quantification of Vascular Pathology in a Chronic Hypoperfusion Model Causing White Matter Damage.

Chronic cerebral hypoperfusion is an important pathological factor in many neurodegenerative diseases, such as cerebral small vessel disease (CSVD). One of the most used animal models for chronic cerebral hypoperfusion is the bilateral common carotid artery stenosis (BCAS) mouse. For the therapy of CSVD and other diseases, it will be beneficial to understand the pathological alterations of the BCAS mouse, particularly vascular pathological changes. A mouse model of BCAS was used, and 8 weeks later, cognitive function of the mice was examined by using novel object recognition test and eight-arm radial maze test. 11.7 T magnetic resonance imaging (MRI) and luxol fast blue staining were used to evaluate the injury of the corpus callosum (CC), anterior commissure (AC), internal capsule (IC), and optic tract (Opt) in the cerebral white matter of mice. Three-dimensional vascular images of the whole brain of mice were acquired using fluorescence micro-optical sectioning tomography (fMOST) with a high resolution of 0.32 × 0.32 × 1.00 μm3. Then, the damaged white matter regions were further extracted to analyze the vessel length density, volume fraction, tortuosity, and the number of vessels of different internal diameters. The mouse cerebral caudal rhinal vein was also extracted and analyzed for its branch number and divergent angle in this study. BCAS modeling for 8 weeks resulted in impaired spatial working memory, reduced brain white matter integrity, and myelin degradation in mice, and CC showed the most severe white matter damage. 3D revascularization of the whole mouse brain showed that the number of large vessels was reduced and the number of small vessels was increased in BCAS mice. Further analysis revealed that the vessel length density and volume fraction in the damaged white matter region of BCAS mice were significantly reduced, and the vascular lesions were most noticeable in the CC. At the same time, the number of small vessels in the above white matter regions was significantly reduced, while the number of microvessels was significantly increased in BCAS mice, and the vascular tortuosity was also significantly increased. In addition, the analysis of caudal rhinal vein extraction revealed that the number of branches and the average divergent angle in BCAS mice were significantly reduced. The BCAS modeling for 8 weeks will lead to vascular lesions in whole brain of mice, and the caudal nasal vein was also damaged, while BCAS mice mainly mitigated the damages by increasing microvessels. What is more, the vascular lesions in white matter of mouse brain can cause white matter damage and spatial working memory deficit. These results provide evidence for the vascular pathological alterations caused by chronic hypoperfusion.

Vacuum-assisted tissue embedding for whole-heart imaging.

The use of combined optical imaging and tissue sectioning has potential for use in visualizing heart-wide fine structures at single-cell resolution. However, existing tissue preparation methods fail to generate ultrathin cavity-containing cardiac tissue slices with minimal deformation. This study developed an efficient vacuum-assisted tissue embedding method to prepare high-filled, agarose-embedded whole-heart tissue. Utilizing optimized vacuum parameters, we achieved 94% filled whole-heart tissue with the thinnest cut slice of 5 µm. We subsequently imaged a whole mouse heart sample using vibratome-integrated fluorescence micro-optical sectioning tomography (fMOST) with a voxel size of 0.32 µm × 0.32 µm × 1 µm. The imaging results indicated that the vacuum-assisted embedding method enabled whole-heart tissue to withstand long-term thin cutting while ensuring that slices were consistent and of high quality.

Whole-brain mapping of histaminergic projections in mouse brain

Histamine is a conserved neuromodulator in mammalian brains and critically involved in many physiological functions. Understanding the precise structure of the histaminergic network is the cornerstone in elucidating its function. Herein, using histidine decarboxylase (HDC)-CreERT2 mice and genetic labeling strategies, we reconstructed a whole-brain three dimensional (3D) structure of histaminergic neurons and their outputs at 0.32 × 0.32 × 2 μm3 pixel resolution with a cutting-edge fluorescence microoptical sectioning tomography system. We quantified the fluorescence density of all brain areas and found that histaminergic fiber density varied significantly among brain regions. The density of histaminergic fiber was positively correlated with the amount of histamine release induced by optogenetic stimulation or physiological aversive stimulation. Lastly, we reconstructed a fine morphological structure of 60 histaminergic neurons via sparse labeling and uncovered the largely heterogeneous projection pattern of individual histaminergic neurons. Collectively, this study reveals an unprecedented whole-brain quantitative analysis of histaminergic projections at the mesoscopic level, providing a foundation for future functional histaminergic study.

Long-range connectome of pyramidal neurons in the sensorimotor cortex

The neocortex mediates information processing through highly organized circuitry that contains various neuron types. Distinct populations of projection neurons in different cortical regions and layers make specific connections and participate in distinct physiological functions. Herein, with the fluorescence micro-optical sectioning tomography (fMOST) and transgenetic mice that targeted intratelencephalic (IT) and pyramidal tract (PT) neurons at specific layers, we dissected the long-range connectome of pyramidal neurons in six subregions of the sensorimtor cortex. The distribution of the input neurons indicated that IT and PT neurons in the same region received information from similar regions, while the neurons in different subregions received from the preferred neuron populations. Both the input and projection areas of these six subregions showed the transverse and longitudinal correspondence in the cortico-cortical, cortico-thalamic, and cortico-striatal circuits, which indicated that the connections were topologically organized. This study provides a comprehensive resource on the anatomical connections of cortical circuits.

Rapid en-bloc hematoxylin-eosin staining for human lung cancer tissue for fluorescence micro-optical sectioning tomography

Objective: To establish a rapid and effective method for en-bloc hematoxylin-eosin (HE) staining and paraffin embedding of human lung cancer and paracancerous tissues which can be applied to fluorescence micro-optical sectioning tomography (fMOST).Methods: Human lung cancer and paracancerous tissues with a size of about 1 cm × 1 cm × 0.3 cm were taken and fixed in 10% neutral formalin. HE staining was performed using a heat water bath to facilitate staining. After staining, isopropyl alcohol was used for dehydration and transparency. Then, 65°C paraffin was used for wax immersion followed by paraffin embedding, while continuous paraffin sections were produced for observation.Results: The tissues stained by en-bloc HE, dehydrated, transparent and wax immersion were slightly smaller in appearance, darker in color and slightly harder in texture than before. After paraffin embedding, the wax blocks did not show any obvious fragmentation, wrinkling or cavity formation, and could be continuously cut into 4-μm thick slices which could be dragged to form wax tapes. The sections could develop flat in waterbath, and the tissues showed no signs of collapse or separation from the paraffin. After sections were picked up and dewaxed, the tissue structure was intact and the cell structure was clear under light microscopy, which could be used to evaluate the pathological features of lung cancer and paracancerous tissues.Conclusion: We propose a suitable en-bloc HE staining of centimeter-sized lung cancer and paracancerous tissues that can be applied to fMOST. It is promising to be used in the accurate identification of structural landmarks and spatial assessment of lung cancer.

Multiscale Analysis of Cellular Composition and Morphology in Intact Cerebral Organoids.

Simple SummaryWe have established a pipeline to analyze the structures of intact millimeter-scale cerebral organoids. By using this pipeline, the morphological and spatial distribution of neurons and GFAP-positive cells in organoids, as well as the spatial distribution of cortical neuron subtypes, were obtained by using fMOST imaging. This study introduced a new approach to monitor cellular composition and morphology of cerebral organoids.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.

Whole-Brain Connectome of GABAergic Neurons in the Mouse Zona Incerta

The zona incerta (ZI) is involved in various functions and may serve as an integrative node of the circuits for global behavioral modulation. However, the long-range connectivity of different sectors in the mouse ZI has not been comprehensively mapped. Here, we obtained whole-brain images of the input and output connections via fluorescence micro-optical sectioning tomography and viral tracing. The principal regions in the input-output circuits of ZI GABAergic neurons were topologically organized. The 3D distribution of cortical inputs showed rostro-caudal correspondence with different ZI sectors, while the projection fibers from ZI sectors were longitudinally organized in the superior colliculus. Clustering results show that the medial and lateral ZI are two different major functional compartments, and they can be further divided into more subdomains based on projection and input connectivity. This study provides a comprehensive anatomical foundation for understanding how the ZI is involved in integrating different information, conveying motivational states, and modulating global behaviors.

Brain-wide mapping of c-Fos expression with fluorescence micro-optical sectioning tomography in a chronic sleep deprivation mouse model

Chronic sleep deprivation (SD) is a common problem for humans and can lead to many deleterious effects, including depression, anxiety, stroke, permanent cognitive deficits, stress, and other physiological diseases. It is vital to acquire information about the relevant neural activities at the whole-brain level to systematically explore the mechanisms of brain dysfunction related to SD. Expression of the immediate-early gene (IEG) Fos in the mouse brain has been widely used as a functional marker of brain activity in the field of neuroscience. However, most previous studies only analyzed the change of c-Fos in several specific brain regions using traditional research methods or in short-term SD model. Here, we applied c-Fos mapping through the fluorescence micro-optical sectioning tomography (fMOST) technique and AAV-PHP.eB to comprehensive analysis the state of cumulative activation across the whole brain in a mouse model of chronic SD. The chronic rapid eyes movement (REM) SD model was induced by moving mice to a separate holding area filled with water. The experimental period lasted for 6 h per day. The results showed that after 14 days of SD, the mice displayed anxiety-like behaviors in open field test and elevated plus maze test, and displayed depression-like behaviors in tail suspension test and the sucrose preference test. The c-Fos + cells were detected in a maximum of 230 brain regions. SD-induced stress model evoked c-Fos expression in several brain regions compared to the control group. In particular, the isocortex-cerebral cortex plate area, including the retrosplenial, anterior cingulate, agranular insular, gustatory, and parasubiculum, appear to be the most sensitive regions after chronic REM SD.

Three-dimensional visualization of heart-wide myocardial architecture and vascular network simultaneously at single-cell resolution.

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.

High-resolution 3D demonstration of regional heterogeneity in the glymphatic system

Accumulating evidence indicates that the glymphatic system has a critical role in maintaining brain homeostasis. However, the detailed anatomy of the glymphatic pathway is not well understood, mostly due to a lack of high spatial resolution 3D visualization. In this study, a fluorescence micro-optical sectioning tomography (fMOST) was used to characterize the glymphatic architecture in the mouse brain. At 30 and 120 min after intracisternal infusion with fluorescent dextran (Dex-3), lectin was injected to stain the cerebral vasculature. Using fMOST, a high-resolution 3D dataset of the brain-wide distribution of Dex-3 was acquired. Combined with fluorescence microscopy and microplate array, the heterogeneous glymphatic flow and the preferential irrigated regions were identified. These cerebral regions containing large-caliber penetrating arteries and/or adjacent to the subarachnoid space had more robust CSF flow compared to other regions. Moreover, the major glymphatic vessels for CSF influx and fluid efflux in the entire brain were shown in 3D. This study demonstrates the regional heterogeneity in the glymphatic system and provides an anatomical resource for further investigation of the glymphatic function.

Hippocampal Mitochondrial Abnormalities Induced the Dendritic Complexity Reduction and Cognitive Decline in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) is a progressive neurodegenerative disease in addition to a traumatic event. Cognitive dysfunction following SCI has been widely reported in patients and animal models. However, the neuroanatomical changes affecting cognitive function after SCI, as well as the mechanisms behind these changes, have so far remained elusive. Herein, we found that SCI accelerates oxidative stress damage of hippocampal neuronal mitochondria. Then, for the first time, we presented a three-dimensional morphological atlas of rat hippocampal neurons generated using a fluorescence Micro-Optical Sectioning Tomography system, a method that accurately identifies the spatial localization of neurons and trace neurites. We showed that the number of dendritic branches and dendritic length was decreased in late stage of SCI. Western blot and transmission electron microscopy analyses also showed a decrease in synaptic communication. In addition, a battery of behavioral tests in these animals revealed hippocampal based cognitive dysfunction, which could be attributed to changes in the dendritic complexity of hippocampal neurons. Taken together, these results suggested that mitochondrial abnormalities in hippocampal neurons induced the dendritic complexity reduction and cognitive decline following SCI. Our study highlights the neuroanatomical basis and importance of mitochondria in brain degeneration following SCI, which might contribute to propose new therapeutic strategies.

Whole-Brain Direct Inputs to and Axonal Projections from Excitatory and Inhibitory Neurons in the Mouse Primary Auditory Area.

Neurons in the primary auditory area (AUDp) innervate multiple brain regions with long-range projections while receiving informative inputs for diverse functions. However, the brain-wide connections of these neurons have not been comprehensively investigated. Here, we simultaneously applied virus-based anterograde and retrograde tracing, labeled the connections of excitatory and inhibitory neurons in the mouse AUDp, and acquired whole-brain information using a dual-channel fluorescence micro-optical sectioning tomography system. Quantified results showed that the two types of neurons received inputs with similar patterns but sent heterogeneous projections to downstream regions. In the isocortex, functionally different areas consistently sent feedback-dominated projections to these neurons, with concomitant laterally-dominated projections from the sensory and limbic cortices to inhibitory neurons. In subcortical regions, the dorsal and medial parts of the non-lemniscal auditory thalamus (AT) were reciprocally connected to the AUDp, while the ventral part contained the most fibers of passage from the excitatory neurons and barely sent projections back, indicating the regional heterogeneity of the AUDp-AT circuit. Our results reveal details of the whole-brain network and provide new insights for further physiological and functional studies of the AUDp.

Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates.

Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are 2 distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. To understand how intramyocardial coronary arteries are generated to connect these 2 coronary vascular populations, we combined genetic lineage tracing, light sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 (methyltransferase-like protein 3) and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.