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

显微镜技术的发展促进了对细胞和生物体如何工作的更复杂的理解。荧光显微镜，允许我们监测单个蛋白质，蛋白质复合物，或细胞器，使用抗体或蛋白质标记，在固定或活的制剂；这反过来又帮助我们将单个蛋白质或蛋白质复合物的行为与其特性的分子和遗传特征联系起来。然而，普通显微镜无法区分距离小于可见光波长一半的物体，也就是大约200-300纳米（百万分之一毫米）。由于细胞机制的许多组成部分之间的距离低于这个极限，了解这种机制是如何工作的需要显微镜技术，甚至可以在这个基本极限以下进行分辨。这种迫切的需求推动了2014年诺贝尔化学奖得主Betzig、Hell和Moerner等人最近开发了许多“超分辨率”显微镜方法。每种方法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，称为受激辐射损耗（STED）显微镜。在这种方法中，激光束利用荧光标签的特性从制备物中产生小于200-300纳米极限的光“像素”——通常为50纳米或更小，这有时足以区分同一蛋白质分子的两端。我们选择了STED，因为它满足了细胞和发育生物学中广泛用户的需求：它允许在所有三个维度上实现超分辨率，能够足够快地实时成像活体制剂，并且允许我们比其他超分辨率方法更深入地成像制剂。我们将把这台显微镜安装在一个允许尽可能广泛的用户使用的环境中。STED现在已经足够成熟，我们可以购买一种商用仪器，它的配置和支持都足够好，允许受过培训的非专业人员使用。专门技术支助、管理委员会、网络预订系统、大量文件、用户培训和用户教育方案将促进广泛使用。我们已经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（包括酵母、果蝇和人类细胞）中基本细胞过程的分子基础。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系统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认识。STED显微镜将如何帮助推进这些项目的例子如下：定位：定位神经元内的膜结构，尤指信号传递的轴突和突触的狭窄空间；并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. 通过研究调节神经元之间突触接触的发展和维持的机制，了解神经元网络是如何形成的。3. 为了了解多功能细胞机器的组织和定位，这些细胞机器可以编排细胞分裂，以实现细胞成分平等地分离成两个子细胞，也可以组织纤毛，既具有运动功能又充当信号天线的细胞产物。4. 了解细胞分裂过程中蛋白质和rna不平等分离的机制，以确保不同细胞类型的正确分化。5. 通过研究核蛋白精确的三维组织来调节基因表达，评估核蛋白功能中的重叠和协同性。

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.