Elucidating how the brain develops from stem cells using revolutionary imaging technologies at single molecule and cell resolutions

最重要的问题和技术差距：了解大脑及其完整的神经元复杂性如何从有限数量的干细胞中发育出来是现代生物学中最重要的问题之一。已知许多动物RNA病毒感染大脑并影响神经干细胞行为，但对其感染机制仍知之甚少。戴维斯实验室一直在使用高度可访问的果蝇模型来解决这些问题，方法是应用新型成像技术直接可视化整个大脑中每个细胞中的所有单个分子转录本（单分子FISH），以及发育中的脑细胞的活细胞成像。参与调节干细胞的大量调节基因为我们理解正常发育和RNA病毒感染的影响提出了一个重大挑战：用现有技术可视化如此大量基因的表达及其突变是不可能的。该项目：我们建议通过利用现有的硬件和软件技术以及Davis实验室与Martin Booth之间的高度跨学科合作来填补这一技术空白，Martin Booth是自适应光学和快速共焦扫描技术的少数世界领导者之一。我们将建立一个负担得起的非常快速的共聚焦显微镜，占地面积小，致力于长期的活细胞成像的大脑在3D中，以及快速的3D成像的多个单分子FISH样品同时进行。一旦建成，显微镜将有许多应用，但最初将主要用于这个高度跨学科的项目。根据学生的背景，工作将涉及光学工程/生物实验/数据分析的平衡。学生将通过Micron Oxford获得世界领先的培训和合作，以获得这三个学科所需的所有相关专业知识。项目的具体目标：1）基于Clarity旋转盘构建定制的无激光共聚焦显微镜，具有前所未有的速度和高通量。2)对已经确定为正确干细胞行为所需的许多关键因素的表型和表达进行系统的实时分析。3)利用该系统在单分子和细胞水平上追踪模式昆虫RNA病毒（Sinvis）在昆虫脑中感染的早期阶段。(ii)该组织的位置（S）将进行研究，注意到所有的iCASE学生都必须与不少于12周的时间的非学术合作伙伴组织进行安置。项目所在地：该项目具有高度的跨学科性。它将以生物化学为基础，但将涉及与工程科学系（距离生物化学5分钟步行路程）的光学和软件工程师的合作。这名学生每年还将在牛津大学的合作公司Aurox Ltd.工作一段时间，(the非学术合作伙伴）共12周。

The overarching question and technological gap: Understanding how the brain and its full neuronal complexity develops from a limited number of stem cells is one of the most important questions in modern biology. A number of animal RNA viruses are known to infect the brain and affect neural stem cell behaviour, but their mechanism of infection is still poorly understood. The Davis lab has been using the highly accessible fruit fly model to address these questions by applying novel imaging technologies for direct visualisation of all the individual molecule transcripts in every cell in a whole brain (single molecule FISH), and live cell imaging of a developing explanted brain. The huge number of regulatory genes involved in regulating the stem cells, presents us with a major challenge for understanding normal development and the effects of RNA virus infections: visualising the expression of such a large number of genes and their mutations is not possible with current technologies. The Project: We propose to fill this technology gap by leveraging existing hardware and software technologies and know-how in a highly interdisciplinary collaboration between the Davis lab and Martin Booth, one of the few world leaders in adaptive optics and rapid confocal scanning technologies. We will build an affordable very fast confocal microscope with a small footprint, dedicated to prolonged live cell imaging of explanted brains in 3D, as well as to rapid 3D imaging of multiple single molecule FISH samples simultaneously. Once built the microscope will have many applications, but will initially be used primarily for this highly interdisciplinary project. Depending on the background of the student, the work will involve a balance of optical engineering / biological experiments / data analysis. The student will have access to world leading training and collaborations through Micron Oxford to all the relevant expertise required for these three disciplines. Specific Aims of the Project: 1) Build a bespoke, laser-free, confocal microscope based on the Clarity spinning disc with unprecedented speed and high throughput capacity. 2) To undertake a systematic live analysis of the phenotypes and expression of numerous key factors already identified as required for correct stem cell behaviour. 3) To use this system to track at the single molecule and cell level the early stages of infection of a model insect RNA virus (Sinvis) in the insect brain. (ii) The location(s) of the organisation(s) in which research will be carried out, noting that all iCASE students are required to undertake a placement with the non-academic partner organisation of no less than twelve-weeks duration. Location of the Project: The project is highly interdisciplinary. It will be based in Biochemistry, but will involve collaborations with optics and software engineers in the Department of Engineering Science (5 minute walk from Biochemistry). The student will also spend some time every year at the collaborating Oxford spinout company, Aurox Ltd., (the non-academic partner) for a total of twelve weeks.