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

通过场发射扫描电子显微镜在背散射电子 (BSE) 成像模式下对脑组织进行高分辨率 3D 重建

• 批准号: BB/I020330/1
• 负责人: Mike Stewart
• 金额: $ 15.28万
• 依托单位: The Open University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI020330%2F1
• 关键词: resolution; reconstructions; brain; tissue; via

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.

我们目前研究的中心问题是促进单个中枢突触功能和修饰的细胞机制。20多年来，我们利用高分辨率透射电子显微镜（TEM）和中枢神经系统海马神经超微结构的三维（3-D）重建，主要是啮齿动物，随后使用了几种学习和可塑性范式。近年来，随着计算机控制的舞台定位的应用，所使用的透射电镜方法取得了长足的进步，而使用胶片捕捉图像已被电子显微镜上高分辨率数码相机的应用所取代。这使得制作数字图像的分辨率至少与胶片衍生的印刷品相当，从而促进了快速获取大量图像。然而，虽然计算能力的进步使图像处理速度更快，但进行3-D重建的过程仍然是一个非常耗时的过程，几乎没有自动化的过程是可行的。在光学显微镜水平上的进步已经提出了研究突触和脊椎之间联系的其他方法，但由于分辨率的限制，这些方法不能精确地测量神经细胞-突触之间的接触。扫描电子显微镜（sem）比tem更适合自动化，可以很容易地处理更大的样品。已经证明，在背散射电子（BSE）成像模式下工作的场发射(FE)- sem与传统制备和染色的TEM切片可以产生与TEM图像相当的优异分辨率和对比度图像。因此，我们的策略将是开发一种新的方法来制作蒙太奇，并重建小鼠或大鼠海马组织的大面积连续切片。我们的研究将利用JEOL UK （SEM和TEM制造商）最先进的设备进行合作。新方法制作如此巨大的蒙太奇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镜（在位置方面）的阶段控制。软件将自动从许多部分的每个部分获取多个图像。每个部分的图像都可以蒙太奇，形成每个部分的单个大蒙太奇，然后可以组合成一个大规模的3d重建。我们相信这种方法可以应用到我们对突触和树突可塑性的常规研究中。

项目成果

Impact of age-related neuroglial cell responses on hippocampal deterioration.

• DOI: 10.3389/fnagi.2015.00057
• 发表时间: 2015
• 期刊: Frontiers in aging neuroscience
• 影响因子: 4.8
• 作者: Ojo JO;Rezaie P;Gabbott PL;Stewart MG
• 通讯作者: Stewart MG

Multiple spine boutons are formed after long-lasting LTP in the awake rat

• DOI: 10.1007/s00429-012-0488-0
• 发表时间: 2012
• 期刊: Brain Structure and Function
• 影响因子: 3.1
• 作者: N. Medvedev;N. Medvedev;G. Dallérac;G. Dallérac;V. I. Popov;V. I. Popov;J. R. Arellano;J. R. Arellano;J. R. Arellano;H. A. Davies;I. Kraev;V. Doyère;Michael G. Stewart

Hippocampal circuit dysfunction in the Tc1 mouse model of Down syndrome.

• DOI: 10.1038/nn.4072
• 发表时间: 2015-09
• 期刊: Nature neuroscience
• 影响因子: 25
• 作者: Witton J;Padmashri R;Zinyuk LE;Popov VI;Kraev I;Line SJ;Jensen TP;Tedoldi A;Cummings DM;Tybulewicz VLJ;Fisher EMC;Bannerman DM;Randall AD;Brown JT;Edwards FA;Rusakov DA;Stewart MG;Jones MW
• 通讯作者: Jones MW

Glia selectively approach synapses on thin dendritic spines.

• DOI: 10.1098/rstb.2014.0047
• 发表时间: 2014-10-19
• 期刊: Philosophical transactions of the Royal Society of London. Series B, Biological sciences
• 作者: Medvedev N;Popov V;Henneberger C;Kraev I;Rusakov DA;Stewart MG
• 通讯作者: Stewart MG

An NCAM mimetic, FGL, alters hippocampal cellular morphometry in young adult (4 month-old) rats.

NCAM 模拟物 FGL 可以改变年轻成年（4 个月大）大鼠的海马细胞形态。

• DOI: 10.1007/s11064-012-0908-9
• 发表时间: 2013
• 期刊: Neurochemical research
• 影响因子: 4.4
• 作者: Ojo B