Pushing Back the Limits of Optical Microscopy - Enhancing dSTORM Resolution through Controlled Fluorophore Switching

视觉使人类能够观察我们周围的世界。看到微小物体的能力，无论是微小的昆虫，蝴蝶翅膀上的彩虹鳞片还是新高清电视上的单个点，都使我们能够直接研究对人类社会有重大影响的微小事物。尽管人类的眼睛很复杂，但它仅限于能够感知小于0.1毫米（或1/万分之一米）的物体。因此，研究小于0.1毫米的物体是不可能的，直到大约1600年发明了高质量的光学和光学显微镜。当时的光学显微镜导致了安东尼·范·列文虎克斯（Antonie van Leeuwenhoeks）对红细胞和微生物（如细菌）的开创性发现。他的发现使光学显微镜在科学家中的普及和使用得到了极大的提高。随后，光学显微镜成为一项关键技术，彻底改变了我们对世界的认识，并为所有现代医学的建立奠定了基础。尽管在设计上有了许多改进，光学显微镜仍然受到物理规则的限制，即光的一个重要特性，即衍射极限。因此，普通光学显微镜只能分辨尺寸大于0.0005毫米（或1/ 200万分之一米）的物体。我们的研究计划涉及一种叫做“超分辨率”光学显微镜的技术。超分辨率显微镜通过结合具有特殊性质的染料分子、激光和计算机图像处理，绕过了衍射极限障碍。这些系统可以看到小到0.00002毫米（1/ 5000万米）的物体，大约是光学显微镜的25倍。使用这些系统将揭示以前未知的细节水平，允许细胞的内部工作。以前所未有的高分辨率呈现。人们只能想象这些新一代的显微镜能揭示出关于生命的什么，以及它们将在未来提供什么样的发现。然而，超分辨率光学显微镜目前是一种高度专业化的工具，只有少数熟练的研究团队使用。我们的目标是提高超分辨率光学显微镜的可及性，使其成为世界各地实验室的标准工具。通过对基础科学的更好理解和实际简单协议的发展，我们将帮助其他研究小组应用超分辨率光学显微镜来理解各种各样的生物系统。我们将使用一种被称为STORM（随机光学重建显微镜）的超分辨率技术，它承诺提供最好的分辨率，同时也是最简单的技术。我们的目标是了解STORM的科学（目前知之甚少），并利用这些知识来简化成像过程和样品制备，从而实现简单，可重复，高分辨率的成像。我们的化学团队将构建新的分子，设计成具有适当性质的专业染料。这些分子染料吸收来自STORM显微镜的激光，然后以一种可以观察到每个单独分子的方式再次释放光。这些单个染料分子发出的光由一台计算机结合起来，逐点构建出我们目标的高分辨率图像。到目前为止，定制染料还不能用于这些系统，而使用的“正常”染料不能提供最佳图像。有了我们定制的染料分子，生物学团队将找出制备生物样品（如细胞、微生物）的最佳方法，以获得最高的分辨率和最佳的可重复性。然后，我们将把我们最好的染料和最好的方案传递给其他团体（商业和公共），使他们能够获得自己目标的最佳高分辨率图像。

Sight allows humans to observe the world around us. The ability to see small objects, whether a minute insect, the iridescent scales on the wings of a butterfly or the individual dots on a new HD television, allows us to directly study tiny things which can have a big influences on human society. Despite the complexity of the human eye, it is limited to being able to perceive objects no bigger than 0.1 millimetres (or 1/10,000th of a metre). Thus study of objects smaller than 0.1 mm was impossible until the invention of high quality optics and the optical microscope, circa 1600. The optical microscopes of the time resulted in the pioneering discoveries of both red blood cells and micro-organisms (e.g. bacteria) by Antonie van Leeuwenhoeks. His discoveries resulted in a massive increase in the popularity and use optical microscopes by scientists. Subsequently the optical microscope has become a key piece of technology, revolutionising our understanding of the world and providing the foundations on which all modern medicine is built.Despite the many design improvements, the optical microscope is still limited by the rules of physics, namely an important property of light known as the diffraction limit. As such, normal optical microscopes can only resolve objects larger than 0.0005 mm (or 1/2,000,000th of a metre) in size. Our research proposal involves a technique called "super-resolution" optical microscopy. Super resolution microscopes circumvent the defraction limit barrier through a combination of dye molecules with special properties, lasers and computerised image processing. These systems can see objects down to 0.00002 mm (1/50,000,000th of a metre) approximately 25 times better than optical microscopy. Use of these systems will reveal previously unknown levels of detail, allowing the internal workings of the cell. to be seen in higher resolution then ever before. One can only imagine what these next generation microscopes could reveal about life and what discoveries they will provide in the future.However super resolution optical microscopy is currently a highly specialist tool, used only by a few skilled research teams. We aim to improve the accessibility of super resolution optical microscopy, allowing it to become a standard tool for laboratories around the world. Though a greater understanding of the underlying science and the development of practically simple protocols we will help other research groups to apply super resolution optical microscopy to understanding a huge variety of biological systems.We will use a super resolution technique known as STORM (STochastic Optical Reconstruction Microscopy) which promises to give the best resolution as well as being the simplest technique to perform. Our aims are to understand the science of STORM (currently poorly understood) and to use that knowledge to simplify both the imaging process and sample preparation, resulting in easy, reproducable, high resolution imaging.Our chemistry team will build new molecules, designed as specialist dyes with just the right properties. These molecular dyes absorb the laser light from the STORM microscope and then release the light again in a way which allows each individual molecule to be observed. The light from these individual dye molecules is combined by a computer to build the high resolution picture of our target point by point. Until now, custom dyes have not been available for use in these systems and "normal" dyes have been used which do not provide the best images. Armed with our custom dye molecules the biology team will then work out the best way to prepare the biological samples (e.g. cells, microbes) to give the highest resolution and the best reproducibility. We will then pass our best dyes and best protocols onto other groups (commercial and public) to allow them to get the best possible high resolution images of their own targets.