High resolution 3-D reconstructions from brain tissue via Field Emission scanning electron microscopy in back scattered electron (BSE) imaging mode

我们目前研究的中心问题是影响单个中央突触的功能和修饰的细胞机制。20多年来，我们一直使用高分辨率电子显微镜(TEM)和三维(3D)重建中枢神经系统(主要是啮齿动物)的海马区的神经超微结构，随后使用了几种学习和可塑性范例。近年来，随着计算机控制舞台定位的应用，所使用的透射电子显微镜方法有了很大的进步，而通过在电子显微镜上应用高分辨率数码相机来取代使用胶片来捕捉图像。这使得能够产生分辨率至少与胶片导出的照片相同的数字图像，从而促进了快速获取数量大得多的图像。然而，尽管计算能力的进步使图像处理变得更加快速，但进行3D重建的过程仍然是一个非常耗时的过程，几乎不可能实现这些过程的自动化。在研究突触和脊椎之间联系的能力方面的替代方法已经被光学显微镜水平的进步所建议，但由于分辨率的限制，这些方法不允许对神经细胞-突触之间的接触进行任何精确的测量。扫描电子显微镜(SEM)比TEM更适合于自动化，并且可以轻松地处理更大的样品。已经证明，在背散射电子(BSE)成像模式下工作的场发射(FE)-扫描电子显微镜(SEMS)与常规制备和染色的透射电子显微镜切片可以产生与透射电子显微镜图像相媲美的良好的分辨率和对比度图像。因此，我们的战略将是开发一种新的方法来制作蒙太奇，并重建大鼠或小鼠海马区组织的大面积连续切片。我们的研究将是一项合作努力，利用JEOL UK(扫描电子显微镜和透射电子显微镜制造商)的最先进设施。制作如此巨大的蒙太奇的新方法的关键是自动图像采集系统，该系统具有非常精确的扫描电子显微镜平台控制(位置方面)。软件将自动从多个部分中的每一个获取多个图像。每个部分的图像可以被剪辑成每个部分的单个大剪辑，然后可以组合成大规模的3-D重建。我们相信，这种方法可以应用于我们对突触和树突可塑性的常规研究。

The central issues of our current research are the cellular mechanisms that contribute to the function and modification of individual central synapses. For over 20 years we have utilised high-resolution transmission electron microscopy (TEM) and 3-dimensional (3-D) reconstructions of neural ultrastructure in the hippocampus of the central nervous system, mainly of rodents, following use a several learning and plasticity paradigms. The TEM methods used have advanced considerably in recent years with application of computer controlled stage positioning, while the use of film to capture images has been supplanted via application of high resolution digital cameras on electron microscopes. This has enabled production of digital images where the resolution is at least the equal of film derived prints and thus has facilitated rapid acquisition of a much greater number of images. However, while advances in computing power have enabled more rapid processing of images, the process of making 3-D reconstructions remains a very time intensive process with little automation of the processes being feasible. Alternative approaches in the ability to study connections between synapses and spines have been suggested by optical advances in at light microscope level but because of resolution limitations these do not permit with any precision, measurements of the contacts between nerve cells - synapses. Scanning electron microscopes (SEMs) are much more suited to automation than TEMs and can easily work with much larger samples. It has already been shown that field emission (FE)-SEMs working in back scattered electron (BSE) imaging mode with conventionally prepared and stained TEM sections can produce excellent resolution and contrast images, comparable to TEM images. Our strategy will therefore be to develop a novel method to make montages, and reconstructions of a large area of serial sections of mouse or rat hippocampal tissue. Our study will be a collaborative effort using state of the art facilities at JEOL UK (SEM and TEM manufacturers). The key of the new method to make such a gigantic montage is the automatic image acquisition system with very accurate stage control of the SEM (in position terms). Software would automatically acquire multiple images from each of a number of sections. Images from each section can be montaged to form single, large montages of each section, which can then be combined into a large-scale 3-D reconstruction. We believe that this methodology can then be applied to our regular studies of synaptic and dendritic plasticity.