Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.
Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.
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成像3D中的乳腺和乳腺肿瘤：光学组织清除和免疫荧光方法。
DOI：
10.1186/s13058-016-0754-9
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发表时间：
2016-12-13
期刊：
影响因子：
--
通讯作者：
Watson CJ
中科院分区：
文献类型：
--
作者：
Lloyd-Lewis B;Davis FM;Harris OB;Hitchcock JR;Lourenco FC;Pasche M;Watson CJ
完整组织的高分辨率3D成像有助于在其天然环境中对复杂结构进行细胞和亚细胞分析。然而，与免疫标记和成像荧光蛋白深入整个器官内的困难限制了它们的应用薄切片或处理的组织制剂，排除了全面和快速的3D可视化。已经建立了几种组织清除方法来规避与不透明标本中的成像深度相关的问题。应用这些技术来研究小鼠乳腺的精细结构还有待研究。对完整的处女乳腺和哺乳期乳腺应用多种组织清除方法，即溶剂清除器官的3D成像，见深部脑（见DB），清除无障碍脑成像鸡尾酒（CUBIC）和被动清晰技术。使用共聚焦，双光子和光片显微镜，他们的兼容性与整体安装免疫荧光标记和乳腺组织的三维成像进行了检查。此外，还评估了其用于分析小鼠乳腺肿瘤的适用性。不同程度的光学透明度，组织保存和荧光信号保存之间观察到不同的清除方法。SeeDB和CUBIC方案分别被认为是体积荧光成像和整体包埋组织化学染色的上级方案。该技术与各种平台上的3D成像兼容，使乳腺导管和小叶-肺泡结构的可视化在透明组织中的深度大大提高。在小鼠乳腺中评估了全器官组织清除方案的效用。大多数方法使用负担得起的和广泛可用的试剂，并与标准的共聚焦显微镜兼容。这些技术能够对乳腺细胞和原位肿瘤进行高分辨率的3D成像和表型分析，并将显着提高我们对正常和病理乳腺发育的理解。本文的在线版本（doi：10.1186/s13058-016-0754-9）包含补充材料，可供授权用户使用。
High-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated. Multiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed. Varying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue. The utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development. The online version of this article (doi:10.1186/s13058-016-0754-9) contains supplementary material, which is available to authorized users.
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DOI：
10.1083/jcb.201004104
发表时间：
2010-05-31
期刊：
The Journal of cell biology
影响因子：
--
作者：
Linkert M;Rueden CT;Allan C;Burel JM;Moore W;Patterson A;Loranger B;Moore J;Neves C;Macdonald D;Tarkowska A;Sticco C;Hill E;Rossner M;Eliceiri KW;Swedlow JR
通讯作者：
Swedlow JR
DOI：
10.1073/pnas.1502264112
发表时间：
2015-05-05
影响因子：
11.1
作者：
Davis, Felicity M.;Janoshazi, Agnes;Putney, James W., Jr.
通讯作者：
Putney, James W., Jr.
平铺 3D 显微图像采集的全局最佳拼接。
DOI：
10.1093/bioinformatics/btp184
发表时间：
2009-06-01
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Preibisch S;Saalfeld S;Tomancak P
通讯作者：
Tomancak P
影响因子：
21.3
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影响因子：
48
作者：
Chung, Kwanghun;Deisseroth, Karl
通讯作者：
Deisseroth, Karl