Bone Research
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2025
·Vol. 13
·期 1
·1-17
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IF 15
Ni Yuehan,Wu Jiamiao,Liu Fengqi,Yi Yating,Meng Xiangjiao,Gao Xiang,Xiao Luyi,Zhou Weiwei,Chen Zexi,Chu Peng,Xing Dan,Yuan Ye,Ding Donghui,Shen Ge,Yang Min,Wu Ronjie,Wang Ling,Melo Luiza Martins Nascentes,Lin Sien,Cheng Xiaoguang,Li Gang,Tasdogan Alpaslan,Ubellacker Jessalyn M.,Zhao Hu,Fang Shentong,Shen Bo
Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional (3D) architecture of tissues and deciphering cellular spatial interactions under physiological and pathological conditions. Deciphering the spatial interaction of leptin receptor-expressing (LepR + ) stromal cells with other compartments in the bone marrow is crucial for a deeper understanding of the stem cell niche and the skeletal tissue. In this study, we introduce an optimized protocol for the 3D analysis of skeletal tissues, enabling the visualization of hematopoietic and stromal cells, especially LepR + stromal cells, within optically cleared bone hemisections. Our method preserves the 3D tissue architecture and is extendable to other hematopoietic sites such as calvaria and vertebrae. The protocol entails tissue fixation, decalcification, and cryosectioning to reveal the marrow cavity. Completed within approximately 12 days, this process yields highly transparent tissues that maintain genetically encoded or antibody-stained fluorescent signals. The bone hemisections are compatible with diverse antibody labeling strategies. Confocal microscopy of these transparent samples allows for qualitative and quantitative image analysis using Aivia or Bitplane Imaris software, assessing a spectrum of parameters. With proper storage, the fluorescent signal in the stained and cleared bone hemisections remains intact for at least 2–3 months. This protocol is robust, straightforward to implement, and highly reproducible, offering a valuable tool for tissue architecture and cellular interaction studies.
研究领域:
细胞生物学
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骨骼学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2025
·Vol. 26
·期 8
·3569
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IF 4.9
Fu Zeng,Lian Huang,Ding Han,Renjie Liu,Kongjia Zhang,Yuwen Su,Tong Su,Yarong Lin,Jianbo Xiu
Recent advances in tissue clearing chemistry have revolutionized three-dimensional imaging by enabling whole-organ antibody labeling, even in thick human tissue samples. However, these techniques face limitations, including reduced clearance efficiency in thick tissue blocks, prolonged processing times, and other challenges specific to formalin-fixed human brain tissue, such as autofluorescence due to the presence of lipofuscin, neuromelanin pigments, and residual blood. To address these challenges, we have developed an improved SHANEL procedure, iSHANEL, which is compatible with brain tissue from multiple species, including nonrodents. iSHANEL significantly enhances the tissue transparency. It effectively removes lipids, particularly sphingolipids, from brain tissue across different species. For brain vasculature imaging, it offers the better visualization of vascular structures at high magnification, reducing light scattering and background noise. Moreover, iSHANEL can be effectively applied to large tissue from pigs and cynomolgus monkeys, and it preserves protein immunogenicity well, as evidenced by immunostaining with the microglia-specific marker IBA-1. We also investigated methods to reduce blood vessel autofluorescence. Overall, iSHANEL provides an improved procedure for the high-resolution 3D imaging of brain tissue across multiple species.
研究领域:
神经科学
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生物化学
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显微成像
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DOI
PubMed
高影响因子
Advanced Science
·
2020
·Vol. 7
·期 8
·1903185
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IF 15.84
Jingtan Zhu,Tingting Yu,Yusha Li,Jianyi Xu,Yisong Qi,Yingtao Yao,Yilin Ma,Peng Wan,Zhilong Chen,Xiangning Li,Hui Gong,Qingming Luo,Dan Zhu
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.
研究领域:
生物医学工程
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神经科学
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病理学
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成像技术
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DOI
PubMed
高影响因子
Biomedical Optics Express
·
2024
·Vol. 15
·期 10
·5609-5624
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IF 2.9
Zizheng Wang,Xiao Xiao,Xiao Xiao,Ziwen Zhou,Yunyin Chen,Tianqi Xia,Xiangyi Sheng,Yiping Han,Wei Gong,Wei Gong,Wei Gong,Ke Si,Ke Si,Ke Si
Many clearing methods achieve high transparency by removing lipid components from tissues, which damages microstructure and limits their application in lipid research. As for methods which preserve lipid, it is difficult to balance transparency, fluorescence preservation and clearing speed. In this study, we propose a rapid water-based clearing method that is fluorescence-friendly and preserves lipid components. FLUID allows for preservation of endogenous fluorescence over 60 days. It shows negligible tissue distortion and is compatible with various types of fluorescent labeling and tissue staining methods. High quality imaging of human brain tissue and compatibility with pathological staining demonstrated the potential of our method for three-dimensional (3D) biopsy and clinical pathological diagnosis.
研究领域:
神经科学
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生物医学工程
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病理学
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DOI
PubMed
高影响因子
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
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2025
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IF 4.7
Jianing Li,Letian He,Wenling Wang,Siyu Wang,Dan Zhang,Liyun Liang,Guangping Song,Yijian Zhang,Shaoqing Yu,Lei Wang,Qiuying Han,Shaoyi Huang,Sen Li,Haiqing Tu,Zengqing Song,Huaibin Hu,Huiyan Li,Yang Yang,Min Wu
As the largest secondary lymphoid organ, the spleen plays a crucial role in initiating and sustaining immune responses against blood-borne pathogens through antigen capture and delivery. It is innervated by both autonomic and sensory nerves, which allows for neural modulation of its immune responses. The intricate spatial structure and precise coordination between immune and neural components is essential for proper splenic function, necessitating three-dimensional (3D) imaging to reveal its architecture. However, the dense fibrous capsule and exceptionally rich vasculature of the spleen pose significant challenges for achieving comprehensive 3D visualization of the entire organ. Here, we systematically evaluated and compared five cutting-edge tissue clearing approaches—ImmuView, FLASH, SHANEL, advanced CUBIC, and Ce3D—for their effectiveness in rendering the spleen transparent for multiplexed antibody staining and high-resolution 3D imaging. Our results indicated that SHANEL provided the clearest visualization of essential splenic neural and immune components. Meanwhile, advanced CUBIC achieved the greatest labeling efficacy for immune cells, albeit with slightly reduced transparency. Importantly, our study marked the first application of these optimized protocols to human spleen tissue, successfully revealing the highly organized immune cell zones and neural networks with enhanced clarity. Notably, we identified the nociceptive sensory innervation within human spleen tissue for the first time. Collectively, these findings establish optimal imaging strategies for visualizing splenic immune cells and neural structure in both murine and human tissues, providing profound insights into the intricate neuroimmune interactions and their pivotal roles in the immune functions of the spleen.
研究领域:
免疫学
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神经科学
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医学影像学
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DOI
PubMed
高影响因子
VIEW
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2025
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IF 8.5
Yi Dai,Jianyi Xu,Jie Hao,Zhang Liu,Chengze Lan,Guanghua Xu,Xizhi Meng,Yating Deng,Peng Wan,Kunxing Liu,Yingtao Yao,Fang Zhao,Peng Fei,Xiu Nie,Gang Lv,Dan Zhu,Tingting Yu
Three-dimensional (3D) visualization and analysis of histological features in human tissue can enhance our comprehension of organ function and elevate the precision of clinical diagnosis. Tissue optical clearing technique has emerged as a potent approach for achieving 3D imaging of large human tissues at a cellular resolution. However, the utilization of tissue clearing to obtain 3D structures of human tissue is hindered by prolonged incubation periods and intricate procedural steps. To address these issues, a rapid and straightforward clearing protocol termed single-step dehydration solvent-based clearing (SSD) is proposed by the introduction of trimethyl borate, which is innovatively incorporated into the tissue clearing process to expedite incubation and streamline the clearing procedure via chemical dehydration. This method reduces the clearing time by 70% and exhibits ideal compatibility with a variety of probes. SSD allows 3D histological imaging and analysis of nerves and vessels, as well as 3D pathological assessment of tumors, within human organs. As an ultrafast clearing alternative, SSD promises to facilitate histological research and enhance clinical diagnostic capabilities.
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DOI
高影响因子
STAR Protocols
·
2023
·
IF 0
Jingtan Zhu,Xiaomei Liu,Jianyi Xu,Zhang Liu,Yating Deng,Junyao Dai,Tingting Yu,Dan Zhu
Summary The majority of fluorescent vessel labeling techniques currently available are limited by their expense, incomplete labeling, or complexity. Here, we present VALID ( v essel l a beling via ge l atin-based l i pophilic d ye solution)—a protocol for complete labeling of different vascular networks. We describe steps for preparing different dye hydrogels, murine vascular casting and tissue harvesting, immunolabeling, tissue clearing, and imaging, as well as detailed analysis of the vascular networks. This protocol is helpful for evaluating vascular lesions in studying different vessel-associated diseases. For complete details on the use and execution of this protocol, please refer to Zhu et al. 1
研究领域:
生物学
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医学成像
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血管生物学
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DOI
PubMed
Scientific Reports
·
2025
·Vol. 15
·期 1
·1-12
·
IF 3.9
Ma Hui,Qin Ruixiu,Yao Qiufeng,Li Yier,Cong Xingshun,Wu Wenhui,Zhao Qi,Ye Hua,Wu Kefeng
Optical transparency methods can facilitate biological tissue optical imaging, which enabled accurate three-dimensional (3D) signal visualization and quantification of complex biological structures. Unfortunately, existing optical clearing approaches present a compromise between maximizing clearing capability, the preservation of fluorescent protein emission and the speed of sample processing. To address this challenge, we synthesis of a 2-hydroxyethyl methacrylate (HEMA)-acrylamide (AAm) copolymer using antipyrine (ATP) and 2,2′-thiodiethanol (TDE) as solvent, which could embed tissue samples rapidly and highly transparent, and compatible with multiple fluorescence labeling. It can enable volumetric imaging of tissue up to the scale of mice organs, shrinkage duration of the clearing and preserve emission from fluorescent proteins and dyes. This copolymer with suitable toughness and plasticity allows the tissue of interest to be sectioned into thin slices, and histological techniques provide high-resolution two-dimensional (2D) images of cells and subcellular structures. Furthermore, HEMA-AAm copolymer -tissue transparent could distinguish cell structures between healthy and diabetic disease in dye-labeled liver tissues, which provides new insights into pathological diagnosis and analysis. Copolymer provides an environment to facilitate high-resolution 3D/2D fluorescence imaging, which enables the study of cellular and tissue morphology in experimental and clinical conditions of interest.
研究领域:
生物医学工程
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荧光成像
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组织光学
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病理诊断
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产品关联
DOI
PubMed
高影响因子
BIOMACROMOLECULES
·
2025
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IF 5.4
Hui Ma,Ruixiu Qin,Qiufeng Yao,Yier Li,Chao Luo,Wenhui Wu,Hui Luo,Qi Zhao,Kefeng Wu
Gel-based tissue clearing technologies represent a significant advancement in three-dimensional imaging. However, the challenge of achieving consistent high resolution across large tissue samples remains an issue. We present a phase-separated eutectogel constructed through radical copolymerization of acrylamide and N,N-dimethylacrylamide in a eutectic solvent (antipyrine/erythritol/imidazole) (AEI/ES) with dynamically tunable polymer–solvent interactions. The prepared eutectogel exhibits high transparency (above 90%) and facilitates rapid clearing of thick tissues and whole organs to a high degree of transparency, while preserving their original size and compatibility with multiple fluorescence. The eutectogel demonstrates high strength (4.2 MPa), favorable Young’s modulus (9.8 MPa), and large toughness (0.78 MJ m–3). The strong mechanical strength of the eutectogel improves tissue strength by more than 120-fold, facilitating the procurement of high-resolution two-dimensional images of cells and subcellular structures. It also offers a wide range of dynamic tunability, thereby providing a universal platform for diverse applications spanning biomedicine to tissue engineering.
研究领域:
生物医学工程
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组织工程
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生物成像
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高分子科学
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材料科学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）