A tridimensional atlas of the developing human head.

The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

Mapping the Fine-Scale Organization and Plasticity of the Brain Vasculature.

The cerebral vasculature is a dense network of arteries, capillaries, and veins. Quantifying variations of the vascular organization across individuals, brain regions, or disease models is challenging. We used immunolabeling and tissue clearing to image the vascular network of adult mouse brains and developed a pipeline to segment terabyte-sized multichannel images from light sheet microscopy, enabling the construction, analysis, and visualization of vascular graphs composed of over 100 million vessel segments. We generated datasets from over 20 mouse brains, with labeled arteries, veins, and capillaries according to their anatomical regions. We characterized the organization of the vascular network across brain regions, highlighting local adaptations and functional correlates. We propose a classification of cortical regions based on the vascular topology. Finally, we analysed brain-wide rearrangements of the vasculature in animal models of congenital deafness and ischemic stroke, revealing that vascular plasticity and remodeling adopt diverging rules in different models.

Deep Learning Reveals Cancer Metastasis and Therapeutic Antibody Targeting in the Entire Body.

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.

Tridimensional Visualization and Analysis of Early Human Development.

Generating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary, and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra- and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be of paramount importance to understanding human development in health and disease. PAPERCLIP.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling.

Mammalian bodies have more than a billion cells per cubic centimeter, which makes whole-body cell (WBC) profiling of an organism one of the ultimate challenges in biology and medicine. Recent advances in tissue-clearing technology have enabled rapid and comprehensive cellular analyses in whole organs and in the whole body by a combination of state-of-the-art technologies of optical imaging and image informatics. In this review, we focus mainly on the chemical principles in currently available techniques for tissue clearing and staining to facilitate our understanding of their underlying mechanisms. Tissue clearing is usually conducted by the following steps: (a) fixation, (b) permeabilization, (c) decolorizing, and (d) refractive index (RI) matching. To phenotype individual cells after tissue clearing, it is important to visualize genetically encoded fluorescent reporters and/or to stain tissues with fluorescent dyes, fluorescent labeled antibodies, or nucleic acid probes. Although some technical challenges remain, the chemical principles in tissue clearing and staining for WBC profiling will enable various applications, such as identifying cellular circuits across multiple organs and measuring their dynamics in stochastic and proliferative cellular processes, for example, autoimmune and malignant neoplastic diseases.

Light-Sheet Microscopy in Neuroscience.

Light-sheet microscopy is an imaging approach that offers unique advantages for a diverse range of neuroscience applications. Unlike point-scanning techniques such as confocal and two-photon microscopy, light-sheet microscopes illuminate an entire plane of tissue, while imaging this plane onto a camera. Although early implementations of light sheet were optimized for longitudinal imaging of embryonic development in small specimens, emerging implementations are capable of capturing light-sheet images in freely moving, unconstrained specimens and even the intact in vivo mammalian brain. Meanwhile, the unique photobleaching and signal-to-noise benefits afforded by light-sheet microscopy's parallelized detection deliver the ability to perform volumetric imaging at much higher speeds than can be achieved using point scanning. This review describes the basic principles and evolution of light-sheet microscopy, followed by perspectives on emerging applications and opportunities for both imaging large, cleared, and expanded neural tissues and high-speed, functional imaging in vivo.

Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

Three-dimensional imaging for the quantification of spatial patterns in microbiota of the intestinal mucosa.

Improving our understanding of host­microbe relationships in the gut requires the ability to both visualize and quantify the spatial organization of microbial communities in their native orientation with the host tissue. We developed a systematic procedure to quantify the three-dimensional (3D) spatial structure of the native mucosal microbiota in any part of the intestines with taxonomic and high spatial resolution. We performed a 3D biogeographical analysis of the microbiota of mouse cecal crypts at different stages of antibiotic exposure. By tracking eubacteria and four dominant bacterial taxa, we found that the colonization of crypts by native bacteria is a dynamic and spatially organized process. Ciprofloxacin treatment drastically reduced bacterial loads and eliminated Muribaculaceae (or all Bacteroidetes entirely) even 10 d after recovery when overall bacterial loads returned to preantibiotic levels. Our 3D quantitative imaging approach revealed that the bacterial colonization of crypts is organized in a spatial pattern that consists of clusters of adjacent colonized crypts that are surrounded by unoccupied crypts, and that this spatial pattern is resistant to the elimination of Muribaculaceae or of all Bacteroidetes by ciprofloxacin. Our approach also revealed that the composition of cecal crypt communities is diverse and that Lactobacilli were found closer to the lumen than Bacteroidetes, Ruminococcaceae, and Lachnospiraceae, regardless of antibiotic exposure. Finally, we found that crypts communities with similar taxonomic composition were physically closer to each other than communities that were taxonomically different.

3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing.

BACKGROUND: Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. RESULTS: We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. CONCLUSIONS: We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

A Cellular Ground Truth to Develop MRI Signatures in Glioma Models by Correlative Light Sheet Microscopy and Atlas-Based Coregistration.

Glioblastoma is the most common malignant primary brain tumor with poor overall survival. Magnetic resonance imaging (MRI) is the main imaging modality for glioblastoma but has inherent shortcomings. The molecular and cellular basis of MR signals is incompletely understood. We established a ground truth-based image analysis platform to coregister MRI and light sheet microscopy (LSM) data to each other and to an anatomic reference atlas for quantification of 20 predefined anatomic subregions. Our pipeline also includes a segmentation and quantification approach for single myeloid cells in entire LSM datasets. This method was applied to three preclinical glioma models in male and female mice (GL261, U87MG, and S24), which exhibit different key features of the human glioma. Multiparametric MR data including T2-weighted sequences, diffusion tensor imaging, T2 and T2* relaxometry were acquired. Following tissue clearing, LSM focused on the analysis of tumor cell density, microvasculature, and innate immune cell infiltration. Correlated analysis revealed differences in quantitative MRI metrics between the tumor-bearing and the contralateral hemisphere. LSM identified tumor subregions that differed in their MRI characteristics, indicating tumor heterogeneity. Interestingly, MRI signatures, defined as unique combinations of different MRI parameters, differed greatly between the models. The direct correlation of MRI and LSM allows an in-depth characterization of preclinical glioma and can be used to decipher the structural, cellular, and, likely, molecular basis of tumoral MRI biomarkers. Our approach may be applied in other preclinical brain tumor or neurologic disease models, and the derived MRI signatures could ultimately inform image interpretation in a clinical setting.SIGNIFICANCE STATEMENT We established a histologic ground truth-based approach for MR image analyses and tested this method in three preclinical glioma models exhibiting different features of glioblastoma. Coregistration of light sheet microscopy to MRI allowed for an evaluation of quantitative MRI data in histologically distinct tumor subregions. Coregistration to a mouse brain atlas enabled a regional comparison of MRI parameters with a histologically informed interpretation of the results. Our approach is transferable to other preclinical models of brain tumors and further neurologic disorders. The method can be used to decipher the structural, cellular, and molecular basis of MRI signal characteristics. Ultimately, information derived from such analyses could strengthen the neuroradiological evaluation of glioblastoma as they enhance the interpretation of MRI data.

Brain-Wide Projections and Differential Encoding of Prefrontal Neuronal Classes Underlying Learned and Innate Threat Avoidance.

To understand how the brain produces behavior, we must elucidate the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) is critical for complex functions including decision-making and mood. mPFC projection neurons collateralize extensively, but the relationships between mPFC neuronal activity and brain-wide connectivity are poorly understood. We performed whole-brain connectivity mapping and fiber photometry to better understand the mPFC circuits that control threat avoidance in male and female mice. Using tissue clearing and light sheet fluorescence microscopy (LSFM), we mapped the brain-wide axon collaterals of populations of mPFC neurons that project to nucleus accumbens (NAc), ventral tegmental area (VTA), or contralateral mPFC (cmPFC). We present DeepTraCE (deep learning-based tracing with combined enhancement), for quantifying bulk-labeled axonal projections in images of cleared tissue, and DeepCOUNT (deep-learning based counting of objects via 3D U-net pixel tagging), for quantifying cell bodies. Anatomical maps produced with DeepTraCE aligned with known axonal projection patterns and revealed class-specific topographic projections within regions. Using TRAP2 mice and DeepCOUNT, we analyzed whole-brain functional connectivity underlying threat avoidance. PL was the most highly connected node with functional connections to subsets of PL-cPL, PL-NAc, and PL-VTA target sites. Using fiber photometry, we found that during threat avoidance, cmPFC and NAc-projectors encoded conditioned stimuli, but only when action was required to avoid threats. mPFC-VTA neurons encoded learned but not innate avoidance behaviors. Together our results present new and optimized approaches for quantitative whole-brain analysis and indicate that anatomically defined classes of mPFC neurons have specialized roles in threat avoidance.SIGNIFICANCE STATEMENT Understanding how the brain produces complex behaviors requires detailed knowledge of the relationships between neuronal connectivity and function. The medial prefrontal cortex (mPFC) plays a key role in learning, mood, and decision-making, including evaluating and responding to threats. mPFC dysfunction is strongly linked to fear, anxiety and mood disorders. Although mPFC circuits are clear therapeutic targets, gaps in our understanding of how they produce cognitive and emotional behaviors prevent us from designing effective interventions. To address this, we developed a high-throughput analysis pipeline for quantifying bulk-labeled fluorescent axons [DeepTraCE (deep learning-based tracing with combined enhancement)] or cell bodies [DeepCOUNT (deep-learning based counting of objects via 3D U-net pixel tagging)] in intact cleared brains. Using DeepTraCE, DeepCOUNT, and fiber photometry, we performed detailed anatomic and functional mapping of mPFC neuronal classes, identifying specialized roles in threat avoidance.

Blueprints from plane to space: outlook of next-generation three-dimensional histopathology.

Here, we summarize the literature relevant to recent advances in three-dimensional (3D) histopathology in relation to clinical oncology, highlighting serial sectioning, tissue clearing, light-sheet microscopy, and digital image analysis with artificial intelligence. We look forward to a future where 3D histopathology expands our understanding of human pathophysiology and improves patient care through cross-disciplinary collaboration and innovation.

Tissue clearing and 3D imaging in developmental biology.

Tissue clearing increases the transparency of late developmental stages and enables deep imaging in fixed organisms. Successful implementation of these methodologies requires a good grasp of sample processing, imaging and the possibilities offered by image analysis. In this Primer, we highlight how tissue clearing can revolutionize the histological analysis of developmental processes and we advise on how to implement effective clearing protocols, imaging strategies and analysis methods for developmental biology.

Concerted morphogenesis of genital ridges and nephric ducts in the mouse captured through whole-embryo imaging.

During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position and spatial relationship with the mesonephric ducts, kidneys and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole-embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements as well as delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis.

In vivo Labeling and Intravital Imaging of Bacterial Infection using a Near-infrared Fluorescent D-Amino Acid Probe.

Bacterial infections still pose a severe threat to public health, necessitating novel tools for real-time analysis of microbial behaviors in living organisms. While genetically engineered strains with fluorescent or luminescent reporters are commonly used in tracking bacteria, their in vivo uses are often limited. Here, we report a near-infrared fluorescent D-amino acid (FDAA) probe, Cy7ADA, for in situ labeling and intravital imaging of bacterial infections in mice. Cy7ADA probe effectively labels various bacteria in vitro and pathogenic Staphylococcus aureus in mice after intraperitoneal injection. Because of Cy7's high tissue penetration and the quick excretion of free probes via urine, real-time visualization of the pathogens in a liver abscess model via intravital confocal microscopy is achieved. The biodistributions, including their intracellular localization within Kupffer cells, are revealed. Monitoring bacterial responses to antibiotics also demonstrates Cy7ADA's capability to reflect the bacterial load dynamics within the host. Furthermore, Cy7ADA facilitates three-dimensional pathogen imaging in tissue-cleared liver samples, showcasing its potential for studying the biogeography of microbes in different organs. Integrating near-infrared FDAA probes with intravital microscopy holds promise for wide applications in studying bacterial infections in vivo.

Imaging plant cell walls using fluorescent stains: The beauty is in the details.

Plants continuously face various environmental stressors throughout their lifetime. To be able to grow and adapt in different environments, they developed specialized tissues that allowed them to maintain a protected yet interconnected body. These tissues undergo specific primary and secondary cell wall modifications that are essential to ensure normal plant growth, adaptation and successful land colonization. The composition of cell walls can vary among different plant species, organs and tissues. The ability to remodel their cell walls is fundamental for plants to be able to cope with multiple biotic and abiotic stressors. A better understanding of the changes taking place in plant cell walls may help identify and develop new strategies as well as tools to enhance plants' survival under environmental stresses or prevent pathogen attack. Since the invention of microscopy, numerous imaging techniques have been developed to determine the composition and dynamics of plant cell walls during normal growth and in response to environmental stimuli. In this review, we discuss the main advances in imaging plant cell walls, with a particular focus on fluorescent stains for different cell wall components and their compatibility with tissue clearing techniques. Lay Description: Plants are continuously subjected to various environmental stresses during their lifespan. They evolved specialized tissues that thrive in different environments, enabling them to maintain a protected yet interconnected body. Such tissues undergo distinct primary and secondary cell wall alterations essential to normal plant growth, their adaptability and successful land colonization. Cell wall composition may differ among various plant species, organs and even tissues. To deal with various biotic and abiotic stresses, plants must have the capacity to remodel their cell walls. Gaining insight into changes that take place in plant cell walls will help identify and create novel tools and strategies to improve plants' ability to withstand environmental challenges. Multiple imaging techniques have been developed since the introduction of microscopy to analyse the composition and dynamics of plant cell walls during growth and in response to environmental changes. Advancements in plant tissue cleaning procedures and their compatibility with cell wall stains have significantly enhanced our ability to perform high-resolution cell wall imaging. At the same time, several factors influence the effectiveness of cleaning and staining plant specimens, as well as the time necessary for the process, including the specimen's size, thickness, tissue complexity and the presence of autofluorescence. In this review, we will discuss the major advances in imaging plant cell walls, with a particular emphasis on fluorescent stains for diverse cell wall components and their compatibility with tissue clearing techniques. We hope that this review will assist readers in selecting the most appropriate stain or combination of stains to highlight specific cell wall components of interest.

The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody.

The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell-specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows.

Multiresolution 3D Optical Mapping of Immune Cell Infiltrates in Mouse Asthmatic Lung.

Asthma is a chronic inflammatory airway disease driven by various infiltrating immune cell types into the lung. Optical microscopy has been used to study immune infiltrates in asthmatic lungs. Confocal laser scanning microscopy (CLSM) identifies the phenotypes and locations of individual immune cells in lung tissue sections by employing high-magnification objectives and multiplex immunofluorescence staining. In contrast, light-sheet fluorescence microscopy (LSFM) can visualize the macroscopic and mesoscopic architecture of whole-mount lung tissues in three dimensions (3D) by adopting an optical tissue-clearing method. Despite each microscopy method producing image data with unique resolution from a tissue sample, CLSM and LSFM have not been applied together because of different tissue-preparation procedures. Here, we introduce a new approach combining LSFM and CLSM into a sequential imaging pipeline. We built a new optical tissue clearing workflow in which the immersion clearing agent can be switched from an organic solvent to an aqueous sugar solution for sequential 3D LSFM and CLSM of mouse lungs. This sequential combination microscopy offered quantitative 3D spatial analyses of the distribution of immune infiltrates in the same mouse asthmatic lung tissue at the organ, tissue, and cell levels. These results show that our method facilitates multiresolution 3D fluorescence microscopy as a new imaging approach providing comprehensive spatial information for a better understanding of inflammatory lung diseases.