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The liver harbors complex cross‐scale structures, and the fibrosis‐related alterations to these structures have a severe impact on the diverse function of the liver. However, the hepatic anatomic structures and their pathological alterations in the whole‐liver scale remain to be elucidated. Combining the micro‐optical sectioning tomography (MOST) system and liver Nissl staining, a first high‐precision whole mouse liver atlas is generated, enabling visualization and analysis of the entire mouse liver. Thus, a detailed 3D panorama of CCl4‐induced liver fibrosis pathology is constructed, capturing the 3D details of the central veins, portal veins, arteries, bile ducts, hepatic sinusoids, and liver cells. Pathological changes, including damaged sinusoids, steatotic hepatocytes, and collagen deposition, are region‐specific and concentrated in the pericentral areas. The quantitative analysis shows a significantly reduced diameter and increased length density of the central vein. Additionally, a deep learning tool is used to segment steatotic hepatocytes, finding that the volume proportion of steatotic regions is similar across liver lobes. Steatosis severity increases with proximity to the central vein, independent of central vein diameter. The approach allows the cross‐scale visualization of multiple structural components in liver research and promotes pathological studies from a 2D to a 3D perspective. Using the MOSTtechnology and whole‐liver Nissl staining method, a multi‐structural 3D pathological panorama of liver fibrosis is obtained. The damaged sinusoids, steatotic hepatocytes, and collagen deposition are all concentrated in pericentral areas. AI segmentation and analysis show that hepatocyte steatosis severity increased with distance from central veins, independent of vein diameter.

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. An approach is established to couple the functional activities with the whole‐brain projection of the same neuron by GCaMP6 sparse labeling and a cross‐modality cell matching. It extends the understanding of structure–function correlations through obtaining functionally relevant projection motifs or projection strengths at the single neuron level.

The effective pulmonary deposition of inhaled particulate carriers loaded with drugs is a prerequisite for therapeutic effects of drug delivery via inhalation route. Revealing the sophisticated lung scaffold and intrapulmonary distribution of particles at three‐dimensional (3D), in‐situ, and single‐particle level remains a fundamental and critical challenge for dry powder inhalation in pre‐clinical research. Here, taking advantage of the micro optical sectioning tomography system, the high‐precision cross‐scale visualization of entire lung anatomy is obtained. Then, co‐localized lung‐wide datasets of both cyto‐architectures and fluorescent particles are collected at full scale with the resolution down to individual particles. The precise spatial distribution pattern reveals the region‐specific distribution and structure‐associated deposition of the inhalable particles in lungs, which is undetected by previous methods. Overall, this research delivers comprehensive and high‐resolution 3D detection of pulmonary drug delivery vectors and provides a novel strategy to evaluate materials distribution for drug delivery. Effective deposition of inhaled drug carriers is a prerequisite for intrapulmonary drug delivery systems. The cross‐scale structures of lungs from alveoli to whole organ and region‐specific distributions of the inhalable particles are revealed with an unprecedented resolution by the fluorescence‐micro optical sectioning tomography and image data mining, which provide a novel strategy to evaluate materials distribution for particulate drug delivery.

Understanding the structural and functional organisation of brain networks is a fundamental objective in neuroscience, with three‐dimensional (3D) reconstruction of single‐neuron morphology serving as a critical foundation. The Golgi staining method, which enables random neuronal labeling and provides high‐contrast signals in both optical and X‐ray microscopy, remains a valuable tool for morphological analysis. However, its widespread application in large‐scale neuronal reconstructions is hindered by signal discontinuities in neuronal branches, high‐density labeling, and complex background interference. While automated reconstruction methods perform well in sparsely labelled and morphologically simple neuronal populations, their effectiveness is limited in Golgi‐stained samples. Here we develop a semi‐automated single‐neuron reconstruction method for Golgi‐stained mouse brain neurons (SNR‐Golgi). By integrating three key technical modules—background denoising, single‐neuron extraction, and branch repair—SNR‐Golgi significantly enhances the accuracy and completeness of neuronal reconstruction. In fluorescence micro‐optical sectioning tomography (fMOST) datasets, SNR‐Golgi demonstrated superior performance in neuronal reconstruction within the mouse somatosensory cortex, achieving a 30% increase in reconstructed branch count, a 76% improvement in total branch length, and a 3.7‐fold increase in axonal length. Additionally, in synchrotron‐based X‐ray imaging datasets, SNR‐Golgi enabled submicron‐resolution 3D reconstruction of single neurons. These results demonstrate that SNR‐Golgi effectively addresses the complexity of Golgi‐stained samples and provides robust technical support for the structural analysis of brain neurons across various imaging modalities.

Revealing neural circuit mechanisms is critical for understanding brain functions. Significant progress in dissecting neural connections has been made using optical imaging with fluorescence labels, especially in dissecting local connections. However, acquiring and tracing brain-wide, long-distance neural circuits at the neurite level remains a substantial challenge. Here, we describe a whole-brain approach to systematically obtaining continuous neuronal pathways in a fluorescent protein transgenic mouse at a one-micron voxel resolution. This goal is achieved by combining a novel resin-embedding method for maintaining fluorescence, an automated fluorescence micro-optical sectioning tomography system for long-term stable imaging, and a digital reconstruction-registration-annotation pipeline for tracing the axonal pathways in the mouse brain. With the unprecedented ability to image a whole mouse brain at a one-micron voxel resolution, the long-distance pathways were traced minutely and without interruption for the first time. With advancing labeling techniques, our method is believed to open an avenue to exploring both local and long-distance neural circuits that are related to brain functions and brain diseases down to the neurite level. ► New device for 3-D imaging of fluorescence mouse brain at 1μmvoxel resolution ► Novel resin-embedding method for transgenic fluorescence labeled mouse brain ► Uninterrupted tracing of brain-wide, long-distance axonal projections ► Revealed several unreported and putative projection pathways in the mouse brain

Systematic cellular and vascular configurations are essential for understanding fundamental brain anatomy and metabolism. We demonstrated a 3D brainwide cellular and vascular (called 3D BrainCV) visualization and quantitative protocol for a whole mouse brain. We developed a modified Nissl staining method that quickly labeled the cells and blood vessels simultaneously in an entire mouse brain. Terabytes 3D datasets of the whole mouse brains, with unprecedented details of both individual cells and blood vessels, including capillaries, were simultaneously imaged at 1-μm voxel resolution using micro-optical sectioning tomography (MOST). For quantitative analysis, we proposed an automatic image-processing pipeline to perform brainwide vectorization and analysis of cells and blood vessels. Six representative brain regions from the cortex to the deep, including FrA, M1, PMBSF, V1, striatum, and amygdala, and six parameters, including cell number density, vascular length density, fractional vascular volume, distance from the cells to the nearest microvessel, microvascular length density, and fractional microvascular volume, had been quantitatively analyzed. The results showed that the proximity of cells to blood vessels was linearly correlated with vascular length density, rather than the cell number density. The 3D BrainCV made overall snapshots of the detailed picture of the whole brain architecture, which could be beneficial for the state comparison of the developing and diseased brain. •Faster brainwide Nissl staining for labeling cells and blood vessels•Terabytes 3D dataset of whole mouse brain with 1mm voxel resolution•Image-processing for brainwide analysis of cellular and vascular configuration.

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.

Background Migraine is a neurological disorder characterized by complex, widespread, and sudden attacks with an unclear pathogenesis, particularly in chronic migraine (CM). Specific brain regions, including the insula, amygdala, thalamus, and cingulate, medial prefrontal, and anterior cingulate cortex, are commonly activated by pain stimuli in patients with CM and animal models. This study employs fluorescence microscopy optical sectioning tomography (fMOST) technology and AAV-PHP.eB whole-brain expression to map activation patterns of brain regions in CM mice, thus enhancing the understanding of CM pathogenesis and suggesting potential treatment targets. Methods By repeatedly administering nitroglycerin (NTG) to induce migraine-like pain in mice, a chronic migraine model (CMM) was established. Olcegepant (OLC) was then used as treatment and its effects on mechanical pain hypersensitivity and brain region activation were observed. All mice underwent mechanical withdrawal threshold, light-aversive, and elevated plus maze tests. Viral injections were administered to the mice one month prior to modelling, and brain samples were collected 2 h after the final NTG/vehicle control injection for whole-brain imaging using fMOST. Results In the NTG-induced CMM, mechanical pain threshold decreased, photophobia, and anxiety-like behavior were observed, and OLC was found to improve these manifestations. fMOST whole-brain imaging results suggest that the isocortex-cerebral cortex plate region, including somatomotor areas (MO), somatosensory areas (SS), and main olfactory bulb (MOB), appears to be the most sensitive area of activation in CM ( P  < 0.05). Other brain regions such as the inferior colliculus (IC) and intermediate reticular nucleus (IRN) were also exhibited significant activation ( P  < 0.05). The improvement in migraine-like symptoms observed with OLC treatment may be related to its effects on these brain regions, particularly SS, MO, ansiform lobule (AN), IC, spinal nucleus of the trigeminal, caudal part (Sp5c), IRN, and parvicellular reticular nucleus (PARN) ( P  < 0.05). Conclusions fMOST whole-brain imaging reveals c-Fos + cells in numerous brain regions. OLC improves migraine-like symptoms by modulating brain activity in some brain regions. This study demonstrates the activation of the specific brain areas in NTG-induced CMM and suggests some regions as a potential treatment mechanism according to OLC. Graphical Abstract

This review explores the application value of fluorescence micro-optical sectioning tomography (fMOST) in diabetes-related cognitive dysfunction research, emphasizing its unique capacity to resolve microstructural alterations in neural circuits and vascular networks, thereby offering novel insights into the pathogenesis of type 2 diabetic cognitive impairment. Existing literature was analyzed to evaluate fMOST's principles and capabilities, including its achievement of whole-brain three-dimensional imaging at sub-micron resolution, simultaneous acquisition of neuronal morphology (soma, dendritic spines, axonal terminals) and vascular networks, and integration with fluorescent labeling to trace prefrontal cortical pyramidal neuron projections under pathological conditions. fMOST technology revealed the critical role of neurovascular coupling dysfunction in diabetic cognitive impairment, demonstrating that interactive damage between neurons and vasculature collectively drives disease progression. In type 2 diabetic models, it identified abnormal synaptic structures in prefrontal/hippocampal pyramidal neurons, vascular network remodeling, and disrupted brain connectivity. Compared to conventional imaging (magnetic resonance imaging/positron emission tomography), fMOST enables concurrent quantitative analysis of synaptic-level neural circuits and microangiopathy, overcoming the resolution limitations of macroscopic imaging. fMOST serves as an indispensable high-precision, multi-scale imaging tool for investigating diabetic cognitive impairment. Future priorities include elucidating dynamic neurovascular unit interactions in diabetic encephalopathy, developing neural circuit-targeted interventions, and advancing interdisciplinary integration to accelerate clinical translation.

Alzheimer’s disease (AD) is an irreversible, neurodegenerative disease that is characterized by memory impairment and executive dysfunction. However, the change of fine structure of neuronal morphology remains unclear in the AD model mouse. In this study, high-resolution mouse brain sectional images were scanned by Micro-Optical Sectioning Tomography (MOST) technology and reconstructed three-dimensionally to obtain the pyramidal neurons. The method of Sholl analysis was performed to analyze the neurons in the brains of 6- and 12-month-old AD mice. The results showed that dendritic complexity was not affected in the entorhinal cortex between 6-month-old mice and 12-month-old mice. The dendritic complexity had increased in the primary motor cortex and CA1 region of hippocampus of 12- month-old mice compared with 6-month-old mice. On the contrary, dendritic complexity in the prefrontal cortex was decreased significantly between 6-month-old and 12-month-old mice. To our knowledge, this is the first study to provide high-resolution brain images of triple transgenic AD mice for statistically analyzing neuronal dendrite complexity by MOST technology to reveal the morphological changes of neurons during AD progression.