Computed tomography of cryogenic biological specimens based on X-ray microscopic images
Computed tomography of cryogenic biological specimens based on X-ray microscopic images
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DOI：
10.1016/s0304-3991(00)00034-6
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发表时间：
2000-08-01
期刊：
影响因子：
2.2
通讯作者：
Schmahl, G
中科院分区：
文献类型：
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作者：
Weiss, D;Schneider, G;Schmahl, G
软X射线显微镜利用水和蛋白质在2.34-4.38 nm波长范围内的光电吸收对比，无需任何染色方法即可显示30 nm大小的蛋白质结构。由于作为X射线物镜的菲涅耳波带片的焦深大，基于X射线显微图像的计算机断层扫描可以用于重建三维样本体积内的局部线性吸收系数。高分辨率X射线图像需要高的样本辐射剂量，并且计算机断层扫描需要在不同视角下拍摄的一系列图像。因此，冷冻显微镜是必要的，以保持水合生物标本的结构完整性，在图像采集。电子储存环BESSY I（柏林）上的低温透射X射线显微镜用于获得冷冻水合的绿色莱茵衣藻的倾斜系列图像。活体标本被插入硼硅酸盐玻璃毛细管中，在第一个实验中。通过浸入液氮中迅速冷却。毛细管样品架允许在180度的全角度范围内进行图像采集。该重建首次显示了冷冻水合生物标本内部60 nm大小的细节，并传达了内部结构的清晰印象。该技术有望广泛应用于细胞核等生物标本。它提供了接近其自然生活状态的水合生物标本的三维结构成像的可能性。(C)2000 Elsevier Science B. V.保留所有权利。
Soft X-ray microscopy employs the photoelectric absorption contrast between water and protein in the 2.34-4.38 nm wavelength region to visualize protein structures down to 30 nm size without any staining methods. Due to the large depth of focus of the Fresnel zone plates used as X-ray objectives, computed tomography based on the X-ray microscopic images can be used to reconstruct the local linear absorption coefficient inside the three-dimensional specimen volume. High-resolution X-ray images require a high specimen radiation dose, and a series of images taken at different viewing angles is needed for computed tomography. Therefore, cryo microscopy is necessary to preserve the structural integrity of hydrated biological specimens during image acquisition. The cryo transmission X-ray microscope at the electron storage ring BESSY I (Berlin) was used to obtain a tilt series of images of the frozen-hydrated green alga Chlamydomonas reinhardtii. The living specimens were inserted into borosilicate glass capillaries and, in this first experiment. rapidly cooled by plunging into liquid nitrogen. The capillary specimen holders allow image acquisition over the full angular range of 180 degrees. The reconstruction shows for the first time details down to 60 nm size inside a frozen-hydrated biological specimen and conveys a clear impression of the internal structures. This technique is expected to be applicable to a wide range of biological specimens, such as the cell nucleus. It offers the possibility of imaging the three-dimensional structure of hydrated biological specimens close to their natural living state. (C) 2000 Elsevier Science B.V. All rights reserved.