Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy.
Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy.
复制标题
DOI：
10.1016/j.bone.2004.10.008
复制
发表时间：
2005-05
期刊：
影响因子：
4.1
通讯作者：
Yasuyo Sugawara;H. Kamioka;T. Honjo;Ken-Ichi Tezuka;T. Takano-Yamamoto
中科院分区：
文献类型：
--
作者：
Yasuyo Sugawara;H. Kamioka;T. Honjo;Ken-Ichi Tezuka;T. Takano-Yamamoto
骨细胞被坚硬的骨基质包围。因此，以前不可能证明骨中骨细胞网络的真实的结构。我们以前报道，它是可能的，观察骨中的骨细胞标记的细胞与荧光和使用共聚焦激光扫描（CLS）显微镜。在这项研究中，我们第一次进行了广泛的三维（3D）骨细胞结构的形态学和形态计量学分析，使用三维重建的荧光图像。用荧光标记的鬼笔环肽对16日龄鸡胚颅骨进行染色，并使用共聚焦激光扫描显微镜进行观察。应用三维重建软件IMARIS、突起长度测量软件NEURON TRACER和细胞表面积/细胞体积分析软件SURPASS对颅骨骨细胞进行形态计量学分析。从IMARIS衍生的图像中，我们发现10个骨细胞的平均值为52.7 ± 5.7个突起，骨细胞中心之间的点对点距离为24.1 ± 2.8 μm。此外，我们可以计算出每个骨细胞平均跨越4180 ± 673 μ m3的骨体积。神经元示踪显示骨细胞突起的长度为0.26 ± 0.02 μm/1 μ m3骨室。此外，SURPASS表明骨细胞的表面积为0.36 ± 0.03 μ m2/1 μ m3骨室，骨细胞胞体与骨室的体积比为9.42% ± 1.18%。总的来说，每个骨细胞的突起的平均总长度、平均表面积和平均体积分别为1070 ± 145 μm、1509 ± 113 μm2和394 ± 49 μm3。从荧光标记的鸡颅骨骨细胞中重建骨细胞网络的真实的结构并获得形态学数据是可能的。
Osteocytes are surrounded by hard bone matrix. Therefore, it has not previously been possible to demonstrate the real architecture of the osteocyte network in bone. We previously reported that it is possible to observe osteocytes in bone by labeling the cells with fluorescence and using confocal laser scanning (CLS) microscopy. In this study, we for the first time conducted an extensive analysis of the morphology and morphometry of the three-dimensional (3D) osteocyte structure using three-dimensionally reconstructed fluorescent images. Sixteen-day-old embryonic chick calvariae were stained with fluorescently labeled phalloidin and observed using a confocal laser scanning microscope. Morphometry of osteocytes in the calvaria was analyzed using extensive three-dimensional reconstructing software IMARIS, process length measuring software NEURON TRACER and cell surface area-/cell volume-analyzing software SURPASS. From the IMARIS-derived images, we found that the average of 10 osteocytes is 52.7 ± 5.7 processes, and the point-to-point distance between centers of the osteocytes was 24.1 ± 2.8 μm. In addition, we could calculate that each osteocyte spans an average of 4180 ± 673 μm3of bone volume. NEURON TRACER showed that the length of osteocyte processes was 0.26 ± 0.02 μm per 1 μm3bone compartment. In addition, SURPASS indicated that the surface area of osteocytes was 0.36 ± 0.03 μm2per 1 μm3bone compartment and that the volume ratio of osteocyte cell body to bone compartment was 9.42% ± 1.18%. Together, the average total length of the processes, the average surface area, and the average volume of one osteocyte were 1070 ± 145 μm, 1509 ± 113 μm2, and 394 ± 49 μm3, respectively. It is possible to reconstruct the real architecture of the osteocyte network and obtain morphometric data from fluorescently labeled osteocytes in chick calvaria.