A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM).
A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM).
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DOI：
10.1038/s41598-017-06942-1
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发表时间：
2017-07-25
影响因子：
4.6
通讯作者：
Usukura J
中科院分区：
文献类型：
--
作者：
Usukura E;Narita A;Yagi A;Sakai N;Uekusa Y;Imaoka Y;Ito S;Usukura J
冷冻切片的使用有助于原子力显微镜(AFM)中组织细胞的形态分析和免疫细胞化学。在用磷酸盐缓冲盐水(PBS)取代包埋介质(蔗糖)后，悬臂可以接触到冰冻切片中的组织样本的所有部分，这种方法已经能够产生一种高分辨率的图像。这些图像类似于冷冻蚀刻复制电子显微镜(EM)获得的图像，而不是来自薄片EM的图像。原子力显微镜图像显示，在EM分辨率下，视杆感光细胞外节(ROS)中的圆盘堆叠并被细胞膜包裹。此外，光镜下可见连接纤毛表面的纤毛项链、突触带的三维构筑以及面向活动部位的突触后膜表面。AFM可以描绘抗视蛋白抗体与结合在盘膜上的二次荧光抗体的分子结合情况。通过与AFM免疫荧光信号的关联，结合共聚焦荧光显微镜，验证了抗视蛋白结合部位的特异性定位。为了证明在视网膜以外的其他组织中的重复性，冰冻切片-AFM也被用来阐明兔腰肌中肌节的分子组织。
The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.
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影响因子：
2.2
作者：
Li, Xinhui;Ji, Tong;Sun, Jielin
通讯作者：
Sun, Jielin
影响因子：
3
作者：
Matsko, N;Mueller, M
通讯作者：
Mueller, M
影响因子：
4.5
作者：
Heuser, J
通讯作者：
Heuser, J
影响因子：
--
作者：
Kodera N;Ando T
通讯作者：
Ando T
影响因子：
2
作者：
Efimov, Anton E.;Tonevitsky, Alexander G.;Matsko, Nadezda B.
通讯作者：
Matsko, Nadezda B.