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首页> 美国卫生研究院文献>other >X-ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-face Imaging for Correlated Light and Electron Microscopy of Biological Specimens
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X-ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

机译：X射线显微镜作为一种方法用于提高生物样品相关光电子显微镜的串行块面成像的准确性和效率

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摘要

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.
机译：最近开发的串行块面扫描电子显微镜（SBEM）的三维电子显微镜（EM）方法已迅速确立其自身作为强大的成像方法的地位。使用这种扫描电子显微镜（SEM）方法进行体积EM成像需要对生物样本进行重金属强烈染色，以提供足够的反向散射电子信号，并使样本具有足够的导电性，以控制充电伪影。与典型的薄层透射电子显微镜相关光和电子显微镜研究相比，这些更极端的重金属染色方案使标本变得不透明，并使跟踪和识别SBEM成像过程的感兴趣区域（ROI）更加困难。我们提出了一种使用X射线显微镜（XRM）来跟踪ROI并提高使用SBEM进行的典型项目所使用的工作流程效率的策略。发现XRM揭示了在SBEM成像中被严重染色的组织中令人印象深刻的细节水平，从而可以识别组织标志物，这些标志物随后可用于指导SEM中的数据收集。此外，可以在XRM体积中检测到使用二氨基联苯胺对单个细胞进行的特异性标记。我们证明，碳化钨颗粒或上转换纳米荧光粉颗粒可以用作基准标记，以进一步提高SBEM成像的精度和效率。

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