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

突破光学显微镜的极限 - 通过受控荧光团切换提高 dSTORM 分辨率

• 批准号: BB/K013971/1
• 负责人: Michael Hall
• 金额: $ 15.19万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK013971%2F1
• 关键词: Pushing; Back; Limits; Optical; Microscopy

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.

视线使人类可以观察我们周围的世界。看到小物体的能力，无论是微小的昆虫，在蝴蝶翅膀上的虹彩鳞片还是在新的高清电视上的单个点上都可以使我们直接研究可能对人类社会产生重大影响的微小事物。尽管人眼的复杂性，但它仅限于感知不超过0.1毫米（或1/10,000米的1/10,000）的物体。因此，直到高质量光学元件和光学显微镜的发明，大约1600的发明是不可能的，这是不可能的。当时的光学显微镜导致了Antonie van Leeuwenhoeks的红细胞和微生物（例如细菌）的开创性发现。他的发现导致流行度大大增加，并使用了科学家的光学显微镜。随后，光学显微镜已成为一项关键技术，彻底改变了我们对世界的理解，并提供了建造所有现代医学的基础。尽管进行了许多设计改进，但光学显微镜仍然受物理规则的限制，即被称为衍射限制的光的重要特性。因此，正常的光学显微镜只能解析大于0.0005 mm（或1/2,000,000米的1/2,000,000米）的物体。我们的研究建议涉及一种称为“超分辨率”光学显微镜的技术。超级分辨率显微镜通过与特殊性能，激光器和计算机图像处理的染料分子的组合来规避脱节极限屏障。这些系统可以看到对象比光学显微镜高出约25倍，低于0.00002毫米（1/50,000,000米的1/50,000,000米）。这些系统的使用将揭示以前未知的细节级别，从而允许细胞的内部工作。以前比以往更高的分辨率可以看到。人们只能想象这些下一代显微镜可以揭示有关生活以及将来将提供的发现。但是，无论超级分辨率光学显微镜是当前是一种高度专业的工具，仅由少数熟练的研究团队使用。我们旨在提高超级分辨率光学显微镜的可及性，从而使其成为世界各地实验室的标准工具。尽管对基本科学和实际简单协议的发展有了更深入的了解，但我们将帮助其他研究小组应用超级分辨率的光学显微镜来理解各种各样的生物系统。我们将使用一种称为Storm的超级分辨率技术（随机光学重建显微镜），可以提供最佳的分辨率，并可以提供最佳的分辨率以及最简单的技术。我们的目的是了解风暴的科学（目前鲜为人知），并使用该知识来简化成像过程和样品制备，从而导致简单，可再现的高分辨率成像。我们的化学团队将建立新的分子，设计为具有正确特性的专业染料。这些分子染料从风暴显微镜中吸收激光光，然后以一种允许观察到每个分子的方式再次释放光。这些单独的染料分子的光被计算机组合在一起，以逐点构建我们目标点的高分辨率图片。到目前为止，尚未在这些系统中使用自定义染料，并且已经使用了“普通”染料，这些染料无法提供最佳图像。随后，用我们的自定义染料分子武装生物学团队将为制定生物样品（例如细胞，微生物）的最佳方法，以提供最高的分辨率和最佳的可重复性。然后，我们将把最好的染料和最佳协议传递给其他群体（商业和公众），以使他们能够获得自己目标的最佳高分辨率图像。

项目成果

Circularly Polarized Luminescence from Helically Chiral N,N,O,O-Boron-Chelated Dipyrromethenes.

• DOI: 10.1002/chem.201504484
• 发表时间: 2016-01-04
• 期刊: Chemistry (Weinheim an der Bergstrasse, Germany)
• 作者: Alnoman RB;Rihn S;O'Connor DC;Black FA;Costello B;Waddell PG;Clegg W;Peacock RD;Herrebout W;Knight JG;Hall MJ
• 通讯作者: Hall MJ