Developing 3D SEM in a broad biological context.
Developing 3D SEM in a broad biological context.
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DOI：
10.1111/jmi.12211
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发表时间：
2015-08
影响因子：
2
通讯作者：
Guérin CJ
中科院分区：
文献类型：
--
作者：
Kremer A;Lippens S;Bartunkova S;Asselbergh B;Blanpain C;Fendrych M;Goossens A;Holt M;Janssens S;Krols M;Larsimont JC;Mc Guire C;Nowack MK;Saelens X;Schertel A;Schepens B;Slezak M;Timmerman V;Theunis C;VAN Brempt R;Visser Y;Guérin CJ
20世纪30年代电子显微镜（EM）问世时，它让科学家们首次得以窥探细胞的纳米世界。在过去的80年里，电子显微镜极大地增进了我们对复杂细胞结构的理解，这些结构是细胞维持生命所需的多种功能的基础。一直以来难以克服的一个缺陷是缺乏体积信息，这主要是由于在透射电子显微镜（TEM）中可观察的切片厚度有限。多年来，科学家们努力通过连续切片重建、TEM断层扫描以及扫描电子显微镜（SEM）技术（如冷冻断裂）来实现三维（3D）电子显微镜成像。尽管每种技术都能提供一些特殊信息，但即使要获取非常小的3D电子显微镜数据集，也需要大量时间和专业知识。大约20年前，科学家们开始利用扫描电子显微镜对包埋组织块进行成像，并在扫描电子显微镜腔室内对这些组织进行连续切片。首先使用聚焦离子束（FIB），随后使用机器人超薄切片机（连续块面，SBF - SEM），显微镜学家能够以能够解决许多重要生物学问题的分辨率收集大量的3D电子显微镜信息，而且效率较高。在此，我们展示一些从我们核心实验室所成像的众多不同样本中获取的3D电子显微镜实例。我们认为，下一步的重大进展将是有效地将利用光学显微镜（LM）获得的功能信息与3D电子显微镜数据集相关联，以便更全面地研究细胞结构与其功能之间的重要联系。
生命是在三维空间中发生的。多年来，先是光学显微镜，然后是电子显微镜，都在努力对细胞的最小部分进行三维成像。随着技术的最新进展以及计算能力的相应提高，科学家们现在能够在纳米尺度上观察细胞的三维世界。在本文中，我们展示了对来自多个物种的多种不同细胞和组织进行高分辨率三维成像的结果。细胞结构的三维重建结果常常表明，与从二维研究中推断的情况相比，它们要复杂得多。将功能性三维光学显微镜研究与三维电子显微镜结果相关联，为我们在理解细胞结构如何与细胞功能相联系方面取得新的进展提供了可能性。
When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions. Life happens in three dimensions. For many years, first light, and then EM struggled to image the smallest parts of cells in 3D. With recent advances in technology and corresponding improvements in computing, scientists can now see the 3D world of the cell at the nanoscale. In this paper we present the results of high resolution 3D imaging in a number of diverse cells and tissues from multiple species. 3D reconstructions of cell structures often revealed them to be significantly more complex when compared to extrapolations made from 2D studies. Correlating functional 3D LM studies with 3D EM results opens up the possibility of making new strides in our understanding of how cell structure is connected to cell function.