Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization.
Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization.
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DOI：
10.1038/nprot.2013.086
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发表时间：
2013
期刊：
影响因子：
14.8
通讯作者：
Kadler KE
中科院分区：
文献类型：
--
作者：
Starborg T;Kalson NS;Lu Y;Mironov A;Cootes TF;Holmes DF;Kadler KE
胶原原纤维是脊椎动物组织中的主要拉伸元件，它们在细胞外基质中以有序束的形式存在。异常的原纤维组装和组织导致瘢痕形成、纤维化、伤口愈合不良和结缔组织疾病。使用透射电子显微镜（TEM）主要通过测量原纤维直径来评估原纤维的形成。在这里，我们描述了一种增强的协议，用于测量原纤维直径以及原纤维体积分数，平均原纤维长度，原纤维横截面形状，和原纤维的三维组织，也是组织功能的主要决定因素。连续切片TEM（ssTEM）已被用于可视化纤维三维组织在体内。然而，连续块面扫描电子显微镜（SBF-SEM）已成为一个时间效率高的替代ssTEM。以下描述的方案适用于制备用于TEM和SBF-SEM（通过3View®）的组织。我们展示了3View®在体内研究胶原纤维组织的能力，并展示了如何找到和跟踪单个纤维。时间范围：从分离组织到获得3D图像堆栈约8天。
Collagen fibrils are the major tensile element in vertebrate tissues where they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess formation of the fibrils, predominantly by measuring fibril diameter. Here we describe an enhanced protocol for measuring fibril diameter as well as fibril-volume-fraction, mean fibril length, fibril cross-sectional shape, and fibril 3D organization that are also major determinants of tissue function. Serial section TEM (ssTEM) has been used to visualize fibril 3D-organization in vivo. However, serial block face-scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View®). We demonstrate the power of 3View® for studying collagen fibril organization in vivo and show how to find and track individual fibrils. Time scale: ~8 days from isolating the tissue to having a 3D image stack.
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影响因子：
3
作者：
Kremer, JR;Mastronarde, DN;McIntosh, JR
通讯作者：
McIntosh, JR
影响因子：
5.6
作者：
Graham, HK;Holmes, DF;Kadler, KE
通讯作者：
Kadler, KE
影响因子：
10.9
作者：
Andres, Bjoern;Koethe, Ullrich;Hamprecht, Fred A.
通讯作者：
Hamprecht, Fred A.
DOI：
10.1083/jcb.103.1.231
发表时间：
1986-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Birk DE;Trelstad RL
通讯作者：
Trelstad RL
影响因子：
3.5
作者：
Michalickova, K;Susic, M;Cole, WG
通讯作者：
Cole, WG