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A multi-user confocal superresolution microscope for cell and developmental biology

用于细胞和发育生物学的多用户共焦超分辨率显微镜

基本信息

批准号：
BB/R000395/1
负责人：
Cahir O'Kane
金额：
$ 72.01万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR000395%2F1
关键词：
multi user confocal superresolution microscope

项目摘要

Developments in microscopy have catalyzed ever more sophisticated understanding of how cells and organisms work. Fluorescence microscopy, allows us to monitor individual proteins, protein complexes, or organelles, using antibodies or protein tagging, in either fixed or live preparations; this in turn helps us link the behavior of individual proteins or protein complexes with molecular and genetic characterization of their properties.However, normal microscopes cannot distinguish between objects that are separated by less than half the wavelength of visible light, or around 200-300 nanometers (millionths of a millimeter). Since many components of the cell machinery are separated by distances below this limit, understanding how this machinery works requires microscopy techniques that can resolve even below this fundamental limit. This compelling need has driven the recent development of a number of "super-resolution" microscopy approaches by (among others) the 2014 Chemistry Nobel Laureates Betzig, Hell and Moerner. Each of these approaches has its own strengths and limitations. We are applying for one type of super-resolution microscope, called a Stimulated Emission Depletion (STED) microscope. In this, laser beams use properties of fluorescent labels to generate "pixels" of light from the preparation that are smaller than the 200-300 nanometer limit - typically 50 nanometers or less, which is sometimes enough to distinguish even the opposite ends of the same protein molecule. We have chosen STED because it meets the needs of a wide range of users in cell and developmental biology: it allows super-resolution in all three dimensions, is fast enough to image live preparations in real time, and allows us to image at greater depths into preparations than other super-resolution methods.We will set up this microscope in an environment that permits its use by as wide a range of users as possible. STED is now mature enough that we can procure a commercially available instrument that is sufficiently well configured and supported to allow use by trained non-specialists. Wide use will be facilitated by specialised technical support, a management committee, a web-based booking system, extensive documentation, user training, and a program of education for users.We already have an active user community who need super-resolution microscopy to understand the molecular basis of fundamental cellular processes in a range of model organisms including yeast, fruitflies, and human cells. These processes represent the core biological functions of the cell, all of which can go awry in a large variety of of long term health problems that include neurological diseases and cancer. Our findings will therefore underpin our knowledge of these diseases. Examples of how a STED microscope will help advance these projects are:1. To localize membrane structures within neurons, especially in the confined spaces of axons and synapses where signals are transmitted; and detecting defects in these structures in fruitflies carrying mutations homologous to human axon degeneration mutations. 2. To understand how neuronal networks form by studying the mechanisms that regulate the development and maintenance of synaptic contacts between identified neurons. 3. To understand the organization and localization of multifunctional cellular machines that can choreograph cell division to achieve equal segregation of cell components into two daughter cells, and that can also organise cilia, cellular outgrowths that can have both motor functions and also act as signalling antennae. 4. To understand mechanisms that achieve unequal segregation of proteins and RNAs during cell division, to ensure correct differentiation of different cell types. 5. To assess overlap and cooperativity in the functions of nuclear proteins through studies of their precise three-dimensional organization to regulate gene expression.

显微镜的发展催生了对细胞和生物体如何工作的更复杂的理解。荧光显微镜，使我们能够监测单个蛋白质、蛋白质复合体或细胞器，使用抗体或蛋白质标记，在固定或活的制剂中；这反过来帮助我们将单个蛋白质或蛋白质复合体的行为与其性质的分子和基因特征联系起来。然而，正常的显微镜无法区分相隔不到可见光波长的一半，或大约200-300纳米(百万分之一毫米)的物体。由于细胞机械的许多部件之间的距离都低于这一限制，因此理解这种机械的工作原理需要显微技术，即使在这一基本限制之下也能分辨出来。这种迫切的需求推动了2014年诺贝尔化学奖获得者贝齐格、地狱和莫纳等人最近开发了一些“超分辨率”显微镜方法。这些方法中的每一种都有自己的优点和局限性。我们正在申请一种超分辨率显微镜，称为受激发射耗尽(STED)显微镜。在这方面，激光束利用荧光标签的特性，从制剂中产生小于200-300纳米限制--通常是50纳米或更少--的“像素”光，这有时甚至足以区分同一蛋白质分子的相反两端。我们选择STED是因为它满足了细胞和发育生物学中广泛用户的需求：它允许所有三个维度的超分辨率，速度足够快，可以实时成像活的制剂，并且允许我们比其他超分辨率方法在更深的地方成像到制剂中。我们将在允许尽可能广泛的用户使用的环境中安装这台显微镜。STED现在已经足够成熟，我们可以获得一种商业上可用的仪器，它的配置和支持足够好，可以供训练有素的非专业人员使用。通过专门的技术支持、管理委员会、基于网络的预订系统、广泛的文档、用户培训和对用户的教育计划，我们将促进广泛的使用。我们已经拥有一个活跃的用户社区，他们需要超分辨率显微镜来了解一系列模式生物中基本细胞过程的分子基础，包括酵母、果蝇和人类细胞。这些过程代表了细胞的核心生物学功能，所有这些功能都可能在包括神经疾病和癌症在内的各种长期健康问题中出错。因此，我们的发现将巩固我们对这些疾病的了解。STED显微镜将如何帮助推进这些项目的例子有：1.定位神经元内的膜结构，特别是在传递信号的轴突和突触的受限空间；以及在携带与人类轴突退化突变同源的突变的果蝇中检测这些结构中的缺陷。2.通过研究神经元之间突触联系的发育和维持机制，了解神经元网络是如何形成的。3.了解多功能细胞机器的组织和定位，这种机器可以编排细胞分裂，实现细胞成分到两个子细胞的平等分离，并且还可以组织纤毛，纤毛是细胞的副产物，既可以具有运动功能，也可以充当信号触角。4.了解细胞分裂过程中蛋白质和RNA不平等分离的机制，确保不同类型细胞的正确分化。5.通过对核蛋白调控基因表达的精确三维组织的研究，评估核蛋白功能的重叠性和协同性。