Counting synapses using FIB/SEM microscopy: a true revolution for ultrastructural volume reconstruction
Counting synapses using FIB/SEM microscopy: a true revolution for ultrastructural volume reconstruction
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DOI：
10.3389/neuro.05.018.2009
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发表时间：
2009-10-05
影响因子：
2.9
通讯作者：
DeFelipe, Javier
中科院分区：
文献类型：
--
作者：
Merchan-Perez, Angel;Rodriguez, Jose-Rodrigo;DeFelipe, Javier
20世纪50年代，透射电子显微镜（TEM）的出现代表了神经回路研究的一个基本步骤。这项技术的应用很快使人们认识到，在正常生活过程中，以及在某些病理或实验环境下，突触的数量会发生变化。从那时起，神经科学的主要目标之一就是定义简单而准确的方法来估计这些变化的幅度。与分析单个剖面相反，透射电镜重建非常耗时和困难。因此，大多数定量研究使用立体学方法来定义在二维空间中研究的突触连接的三维特征。在这里，为了精确计算每单位体积的突触数量，我们应用了一种新的三维重建方法，该方法涉及聚焦离子束铣削和扫描电子显微镜（FIB/SEM）的结合。我们发现FIB/SEM获得的图像与TEM获得的图像相似，但FIB/SEM的优点是可以快速自动地生成大量组织的序列重建。此外，我们比较了用立体学方法得到的突触数量估定值与FIB/SEM重建得到的值。我们得出结论，FIB/SEM不仅提供了每体积突触的实际数量，而且比其他现有的TEM方法更容易和更快地使用。更重要的是，它还避免了大多数由立体方法引入的错误，并克服了与这些技术相关的困难。
The advent of transmission electron microscopy (TEM) in the 1950s represented a fundamental step in the study of neuronal circuits. The application of this technique soon led to the realization that the number of synapses changes during the course of normal life, as well as under certain pathological or experimental circumstances. Since then, one of the main goals in neurosciences has been to define simple and accurate methods to estimate the magnitude of these changes. Contrary to analysing single sections, TEM reconstructions are extremely time-consuming and difficult. Therefore, most quantitative studies use stereological methods to define the three-dimensional characteristics of synaptic junctions that are studied in two dimensions. Here, to count the exact number of synapses per unit of volume we have applied a new three-dimensional reconstruction method that involves the combination of focused ion beam milling and scanning electron microscopy (FIB/SEM). We show that the images obtained with FIB/SEM are similar to those obtained with TEM, but with the advantage that FIB/SEM permits serial reconstructions of large volumes of tissue to be generated rapidly and automatically. Furthermore, we compared the estimates of the number of synapses obtained with stereological methods with the values obtained by FIB/SEM reconstructions. We concluded that FIB/SEM not only provides the actual number of synapses per volume but it is also much easier and faster to use than other currently available TEM methods. More importantly, it also avoids most of the errors introduced by stereological methods and overcomes the difficulties associated with these techniques.