Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples.
Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples.
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DOI：
10.3791/62045
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发表时间：
2021-03-26
期刊：
影响因子：
--
通讯作者：
Burns AR
中科院分区：
文献类型：
--
作者：
Courson JA;Landry PT;Do T;Spehlmann E;Lafontant PJ;Patel N;Rumbaut RE;Burns AR
连续块面扫描电子显微镜（SBF-SEM）允许收集数百到数千个连续配准的超微结构图像，提供前所未有的组织显微解剖的三维视图。虽然近年来SBF-SEM的使用呈指数级增长，但适当的组织制备和成像参数等技术方面对于这种成像模式的成功至关重要。该成像系统得益于设备的自动化特性，允许在成像过程中无人值守显微镜，并在一天内自动收集数百张图像。然而，如果没有适当的组织制备，细胞超微结构可能会改变，从而可能得出错误或误导性的结论。此外，通过扫描树脂包埋的生物样品的块面来生成图像，这通常提出了必须解决的挑战和考虑因素。在成像过程中，电子在块内的积累，称为“组织充电”，可能导致对比度损失和无法识别细胞结构。此外，虽然增加电子束强度/电压或降低束扫描速度可以增加图像分辨率，但这也可能具有损坏树脂块和使成像系列中的后续图像失真的不利副作用。在这里，我们提出了一个常规协议的生物组织样品的制备，保留细胞超微结构和减少组织充电。我们还提供成像考虑因素，以快速获取高质量的序列图像，对组织块的损伤最小。该协议概述了一个常规的方法，使用连续块面扫描电子显微镜（SBF-SEM），一个强大的3D成像技术。SBF-SEM的成功应用取决于适当的固定和组织染色技术，以及仔细考虑成像设置。该协议包含了对整个过程的实际考虑。
Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as “tissue charging,” can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block. This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.
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影响因子：
4.6
作者：
He, Q.;Hsueh, M.;Leapman, R. D.
通讯作者：
Leapman, R. D.
影响因子：
--
作者：
Katoh K
通讯作者：
Katoh K
DOI：
10.3791/3564
发表时间：
2012-07-30
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Gage, Gregory J;Kipke, Daryl R;Shain, William
通讯作者：
Shain, William
影响因子：
9.8
作者：
Belevich I;Joensuu M;Kumar D;Vihinen H;Jokitalo E
通讯作者：
Jokitalo E
影响因子：
3.7
作者：
Courson, Justin A.;Smith, Ian;Burns, Alan R.
通讯作者：
Burns, Alan R.