Three-dimensional visualization of human brain tumors using the CUBIC technique.

Application of tissue clearing techniques on human brain tumors is still limited. This study was to investigate the application of CUBIC on 3D pathological studies of human brain tumors. Brain tumor specimens derived from 21 patients were cleared with CUBIC. Immunostaining was conducted on cleared specimens to label astrocytes, microglia and microvessels, respectively. All tumor specimens achieved transparency after clearing. Immunostaining and CUBIC are well compatible in a variety of human brain tumors. Spatial morphologies of microvessels, astrocytes and microglia of tumors were clearly visualized in 3D, and their 3D morphological parameters were easily quantified. By comparing the quantitative morphological parameters of microvessels among brain tumors of different malignancy, we found that mean vascular diameter was positively correlated with tumor malignancy. Our study demonstrates that CUBIC can be successfully applied to 3D pathological studies of various human brain tumors, and 3D studies of human brain tumors hold great promise in helping us better understand brain tumor pathology in the future.

Perspective: Extending the Utility of Three-Dimensional Organoids by Tissue Clearing Technologies.

An organoid, a self-organizing organ-like tissue developed from stem cells, can exhibit a miniaturized three-dimensional (3D) structure and part of the physiological functions of the original organ. Due to the reproducibility of tissue complexity and ease of handling, organoids have replaced real organs and animals for a variety of uses, such as investigations of the mechanisms of organogenesis and disease onset, and screening of drug effects and/or toxicity. The recent advent of tissue clearing and 3D imaging techniques have great potential contributions to organoid studies by allowing the collection and analysis of 3D images of whole organoids with a reasonable throughput and thus can expand the means of examining the 3D architecture, cellular components, and variability among organoids. Genetic and histological cell-labeling methods, together with organoid clearing, also allow visualization of critical structures and cellular components within organoids. The collected 3D data may enable image analysis to quantitatively assess structures within organoids and sensitively/effectively detect abnormalities caused by perturbations. These capabilities of tissue/organoid clearing and 3D imaging techniques not only extend the utility of organoids in basic biology but can also be applied for quality control of clinical organoid production and large-scale drug screening.

Tissue clearing technique: Recent progress and biomedical applications.

Organisms are inherently three dimensional, thus comprehensive understanding of the complicated biological system requires analysis of organs or even whole bodies in the context of three dimensions. However, this is a tremendous task since the biological specimens are naturally opaque, a major obstacle in whole-body and whole-organ imaging. Tissue clearing technique provides a prospective solution and has become a powerful tool for three-dimensional imaging and quantification of organisms. Tissue clearing technique aims to make tissue transparent by minimizing light scattering and light absorption, thus allowing deep imaging of large volume samples. When combined with diverse molecular labeling methods and high-throughput optical sectioning microscopes, tissue clearing technique enables whole-body and whole-organ imaging at cellular or subcellular resolution, providing detailed and comprehensive information about the intact biological systems. Here, we give an overview of recent progress and biomedical applications of tissue clearing technique. We introduce the mechanisms and basic principles of tissue clearing, and summarize the current tissue clearing methods. Moreover, the available imaging techniques and software packages for data processing are also presented. Finally, we introduce the recent advances in applications of tissue clearing in biomedical fields. Tissue clearing contributes to the investigation of structure-function relationships in intact mammalian organs, and opens new avenues for cellular and molecular mapping of intact human organs. We hope this review contributes to a better understanding of tissue clearing technique and can help researchers to select the best-suited clearing protocol for their experiments.

Comparative Analyses of Clearing Efficacies of Tissue Clearing Protocols by Using a Punching Assisted Clarity Analysis.

The advent of tissue clearing methods, in conjunction with novel high-resolution imaging techniques, has enabled the visualization of three-dimensional structures with unprecedented depth and detail. Although a variety of clearing protocols have been developed, little has been done to quantify their efficacies in a systematic, reproducible fashion. Here, we present two simple assays, Punching-Assisted Clarity Analysis (PACA)-Light and PACA-Glow, which use easily accessible spectroscopy and gel documentation systems to quantify the transparency of multiple cleared tissues simultaneously. We demonstrate the use of PACA-Light and PACA-Glow to compare twenty-eight tissue clearing protocols on rodent brains. We also show that regional differences exist in tissue transparency in the rodent brain, with cerebellar tissue consistently achieving lower clearing levels compared to the prefrontal or cerebral cortex across all protocols. This represents the largest comparative study of tissue clearing protocols to date, made possible by the high-throughput nature of our PACA platforms.

Applications of tissue clearing in the spinal cord.

The locomotor networks in the spinal cord coordinate muscle contractions and govern limb movements, ensuring postural body stability and smooth locomotion at different speeds. Dissecting the organization and connection of spinal motor networks is crucial to interpret their functional roles and thus can help to design more specific interventions for motor system diseases. Traditional two-dimensional (2D) histological sectioning is inadequate to accurately dissect complex architecture of the spinal cord as it provides only partial spatial information about spinal neural circuits. It is particularly difficult for quantitative analysis of axon regeneration after injury with 2D tissue sections, because it shows axonal fragments rather than spatial trajectory of regenerating axons. Therefore, three-dimensional (3D) imaging and analysis are extremely necessary for investigations of spinal cord structure and function. Although 3D spatial structure of the spinal cord can be reconstructed by serial sectioning, this approach is laborious and prone to image distortion. The recently emerging tissue clearing technique enables 3D imaging of the entire spinal cord at cellular resolution without tissue sectioning. The development of tissue clearing contributes to revealing the organization and function of spinal circuits and elucidating associated mechanisms underlying certain behaviours in health and disease. In this paper, we give an overview of the current clearing methods and introduce available labelling and imaging techniques as well as data processing software. Finally, we demonstrate the recent applications of tissue clearing in the spinal cord. Tissue clearing technique provides a novel tool for 3D imaging and quantification of the spinal cord, and benefits investigations of structure-function relationship of spinal networks. This review might help researchers to find the potential of tissue clearing in the studies of spinal cord and select appropriate clearing protocol for their experimental schemes.

Pipe-3D: A Pipeline Based on Immunofluorescence, 3D Confocal Imaging, Reconstructions, and Morphometry for Biliary Network Analysis in Cholestasis.

Cholestasis, the impairment of bile flux out of the liver, is a common complication of many pathological liver disorders, such as cholangiopathies, primary biliary sclerosis, and primary biliary cirrhosis. Besides accumulation of bile acids in the liver and blood, it leads to a proliferative response of the biliary tree termed as a ductular reaction. The ductular reaction is characterized by enhanced proliferation of cholangiocytes, which form the epithelial lining of bile ducts. This strong reaction of the biliary tree has been reported to generate a source of progenitor cells that can differentiate to hepatocytes or cholangiocytes during regeneration. On the other hand, it can cause periportal fibrosis eventually progressing to cirrhosis and death. In 2D histology, this leads to the appearance of an increased number of duct lumina per area of tissue. Yet, the biliary tree is a 3D vstructure and the appearance of lumina in thin slices may be explained by the appearance of novel ducts or by ramification or convolution of existing ducts in 3D. In many such aspects, traditional 2D histology on thin slices limits our understanding of the response of the biliary tree. A comprehensive understanding of architecture remodeling of the biliary network in cholestasis depends on robust 3D sample preparation and analysis methods. To that end, we describe pipe-3D, a processing and analysis pipeline visualization based on immunofluorescence, confocal imaging, surface reconstructions, and automated morphometry of the biliary network in 3D at subcellular resolution. This pipeline has been used to discover extensive remodeling of interlobular bile ducts in cholestasis, wherein elongation, branching, and looping create a dense ductular mesh around the portal vein branch. Surface reconstructions generated by Pipe-3D from confocal data also show an approximately fivefold enhancement of the luminal duct surface through corrugation of the epithelial lamina, which may increase bile reabsorption and alleviate cholestasis. The response of interlobular ducts in cholestasis was shown to be in sharp contrast to that of large bile ducts, de novo duct formation during embryogenesis. It is also distinct from ductular response in other models of hepatic injury such as choline-deficient, ethionine-supplemented diet, where parenchymal tissue invasion by ducts and their branches is observed. Pipe-3D is applicable to any model of liver injury, and optionally integrates tissue clearing techniques for 3D analysis of thick (>500 µm) tissue sections.

Label-free optical projection tomography for quantitative three-dimensional anatomy of mouse embryo.

Recent progress in three-dimensional optical imaging techniques allows visualization of many comprehensive biological specimens. Optical clearing methods provide volumetric and quantitative information by overcoming the limited depth of light due to scattering. However, current imaging technologies mostly rely on the synthetic or genetic fluorescent labels, thus limits its application to whole-body visualization of generic mouse models. Here, we report a label-free optical projection tomography (LF-OPT) technique for quantitative whole mouse embryo imaging. LF-OPT is based on the attenuation contrast of light rather than fluorescence, and it utilizes projection imaging technique similar to computed tomography for visualizing the volumetric structure. We demonstrate this with a collection of mouse embryo morphologies in different stages using LF-OPT. Additionally, we extract quantitative organ information applicable toward high-throughput phenotype screening. Our results indicate that LF-OPT can provide multi-scale morphological information in various tissues including bone, which can be difficult in conventional optical imaging technique.

Spatial impact of microglial distribution on dynamics of dendritic spines.

Microglia regulate synapse stability and remodeling through multiple molecular pathways. Regulated spatial distribution of microglia within nervous tissues may affect synapse dynamics. Here, we focused on the spatial relationship between microglia and spine synapses in the mouse neocortex and found that the distance between microglial cell bodies (MCBs) and spines is a critical parameter in spine stability. The region close to MCBs contains microglial processes with higher density and with more spine contacts. This region also shows more extensive exploration of tissue space by microglial processes. To test if the relative positions between MCBs and spines are important for spine stability, we simultaneously imaged spines and microglia in vivo and found negative correlation between spine-MCB distance and spine stability. Optical clearing methods enabled us to record the positions of all microglia in a large cortical volume and indicated their mutually exclusive distribution with similar density across cortical layers. This spatial arrangement of microglia is responsible for the repeated appearance of domains close to MCBs along dendritic arborization. The microglial position was largely independent of other cellular components. These results suggest that the spatial arrangement of microglia is critical for generating repetitive domains of synaptic instability along dendrites, which operates independently of other glial components.