Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies
Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies
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DOI：
10.1016/j.bpj.2014.09.011
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发表时间：
2014-10-21
影响因子：
3.4
通讯作者：
Larabell, Carolyn A.
中科院分区：
文献类型：
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作者：
Smith, Elizabeth A.;McDermott, Gerry;Larabell, Carolyn A.
软X射线断层扫描（SXT）越来越多地被认为是一种有价值的方法，可视化和量化的冷冻保存细胞的超微结构。在这里，我们描述了SXT与低温共焦荧光断层扫描（CFT）的组合。这种相关的方法允许将分子定位数据，各向同性的精度，到高分辨率的三维（3-D）SXT重建的细胞。CFT数据采集首先使用低温适应共焦光学显微镜，其中的标本是耦合到一个高数值孔径物镜透镜的浸没流体。然后将样品冷冻转移到软X射线显微镜（SXM）进行SXT数据采集。在两种类型的数据中可见的基准标记作为共同的地标，使两个互补的断层重建的准确的共对准。我们使用这种方法来鉴定女性v-abl转化的胸腺淋巴瘤细胞中的失活X染色体（Xi），通过用CFT定位增强的绿色荧光蛋白标记的macroH 2A。分子定位数据用于指导SXT重建中Xi的分割，允许表征近天然状态细胞中的Xi拓扑排列。习近平被认为采用了许多不同的拓扑结构，没有特定的安排占主导地位。
Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.