Life in 3D, expanding the structural length scales via Serial Block Face Scanning Electron Microscopy (SBF-SEM)

生物成像是生命科学研究的关键技术之一，但研究生命功能的方式往往是通过将其减少到二维投影，例如通过光学和/或电子显微镜。尽管这种方法已经并将在未来带来突破性的发现，但人们意识到，如果可能的话，应该尽可能多地在3D环境中研究“生命”，理想的情况是在组织或有机体的背景下。因此，一些较新的显微技术聚焦于这一方面，并允许在所有3个维度上成像大体积，例如通过使用光片光学显微镜。电子显微镜通常专注于更高分辨率的结构信息，但除了结构低温电子显微镜(EM)揭示更多分子细节的“分辨率革命”之外，在体积EM方面也发生了一场更安静但可能同样重要的革命。通过顺序扫描嵌入样品的块面并去除扫描的顶层，可以获得大的高分辨率三维体积。虽然分辨率稍低，但这个体积比标准(连续厚层)电子断层扫描的可能体积要大得多。这允许完整细胞的结构可视化，以及这些细胞与其他细胞或组织内的细胞外基质的相互作用。由于所有的生命都是建立在三维空间中的，序列块面扫描电子显微镜(SBF-SEM)现在已经成为越来越多的生命科学研究的选择方法。我们想申请一种集成了钻石刀基切片机的扫描电子显微镜(SEM)。我们将把这项技术集成到沃尔夫森生物成像设备中，这是布里斯托尔大学生物医学系的中央显微镜设备，其中包括光学和电子显微镜，并以相关的显微镜技术而闻名。我们将应用该工具来研究各种只能以3D方式研究的研究问题。这些包括但当然不限于大脑中突触的形成，巨噬细胞对组织的渗透，以及血液中血小板的形成和释放。重要的是，这个工具还将使我们能够研究细胞外基质与细胞的相互作用，这是3D成像至关重要的一个领域。最后但同样重要的是，我们将使用显微镜来促进我们对合成生物学领域的理解，合成生物学领域是BBSRC的亮点领域之一。布里斯托尔的一些合成生物学研究集中在人造细胞外支架的开发上，其想法是在上面生长细胞，并最终将其用于移植。为了研究种子细胞与这种基质载体的相互作用，SBFSEM将是至关重要的。培养下一代科学家是沃尔夫森生物成像设施的一个重要方面。作为国际EMBO课程和其他国家培训课程的发源地，这项技术将特别有兴趣无缝地整合到其中一些培训课程中，为学生提供最先进技术的培训。

Bioimaging is one of the key technologies for life science research but the way life functions has however often been studied by reducing this to 2-dimensional projections for instance by light and / or electron microscopy. Although this approach has led and will also in the future lead to breakthrough discoveries there is a realisation that if possible "life" should be studied as much as possible in a 3D environment, ideally in the context of a tissue or organism. Some of the newer microscopy techniques therefore are focussing on this aspect and allow for imaging large volumes in all 3 dimensions, e.g. by using light sheet light microscopy. Electron Microscopy has generally focussed on higher resolution structural information but alongside the "resolution revolution" in structural cryo Electron Microscopy (EM) which is revealing ever greater molecular details, there has been a quieter but probably equally important revolution in volume EM. By sequentially scanning the block face of an embedded sample and removing the scanned top layer, a large high-resolution 3-dimensional volume can be acquired. Although at somewhat lower resolution this volume is much larger than possible by standard (serial thick section) Electron Tomography. This allows for the structural visualisation of complete cells and the interactions of these cells with other cells or the extracellular matrix inside a tissue. As all life is built in 3 Dimensions, Serial Block Face Scanning Electron Microscopy (SBF-SEM) has now become the method of choice for a growing number of life science studies.We would like to apply for a Scanning Electron Microscope (SEM) with an integrated diamond knife based microtome. We will integrate the technology into the Wolfson Bioimaging Facility, the centralised microscopy facility of the Faculty of Biomedical Sciences at the University of Bristol which houses both light and electron microscopy and is well-known for its Correlative Microscopy technologies.We will apply the tool to study a variety of research questions that can only be studied in 3D. These include but are certainly not limited to the formation of synapses in the brain, the infiltration of macrophages into tissue, and the formation and release of platelets in the blood stream. Importantly this tool will allow us to also study the interaction of the extracellular matrix with cells, an area where 3D imaging is critical.Last but not least we will use the microscope to advance our understanding in the Synthetic Biology field, one of the highlight areas of the BBSRC. Some of the Synthetic Biology research in Bristol focusses on the development of artificial extracellular supports with the idea to grow cells on, and ultimately to use these for transplantation. In order to study the interaction of seeded cells with such matrix supports the SBFSEM will be of critical importance.Training of the next generation of scientists is an important aspect within the Wolfson Bioimaging Facility. As home of an international EMBO course and other national training courses the technology would be of special interest to seamlessly integrate into some of those training courses, providing students with training in the latest state of the art technology.