Non-perturbative use of fluorescent proteins for super-resolution microscopy

荧光显微镜可以对活细胞中的现象进行动态观察。然而，直到最近，光的波状性质及其相关的衍射，将光学显微镜的分辨率限制在~200纳米。新的“超分辨率”荧光显微镜方法已经开发出来，以克服“传统”显微镜的衍射极限。超分辨率显微镜方法的巨大影响被其发明者认可，并被授予诺贝尔化学奖（2014年）。超分辨率方法可以将荧光显微镜的分辨率提高到~5 nm，从而可以非常详细地观察分子过程。尽管光学方法取得了这些进步，但标记感兴趣的生物分子仍然是一个挑战，特别是在活细胞内。将感兴趣的蛋白质直接融合到荧光蛋白是一种常见的策略，因为它是遗传可编码的，可以用于活细胞成像，并且适合超分辨率显微镜（通过明智地选择荧光蛋白）。不幸的是，荧光蛋白非常大（约27 kDa），直接融合会干扰目标蛋白的功能、组装和/或位置。为了解决这些问题，学生将开发一种新的超分辨率成像方法，该方法基于完全遗传可编码的标签策略，不需要使用外源底物，并为感兴趣的生物分子添加最小破坏性标签。而不是将荧光蛋白本身融合到感兴趣的蛋白质上，一个短肽标签将被遗传编码到它上面。然后，该标签可以以共价或非共价方式连接到同样在细胞内以受控方式表达的伴侣蛋白上。这种方法已被证明可以很好地标记细菌和酵母中的蛋白质1,2。学生将在这项工作的基础上，将该策略应用于哺乳动物细胞。最重要的是，他们将使用光切换荧光蛋白mEoS开发这种方法，这将使使用光激活定位显微镜生成超分辨率图像成为可能。我们特别感兴趣的是应用这种新策略来可视化人类来源的神经元中形成的淀粉样蛋白。培训：该项目的主管将结合他们在蛋白质结构和设计（Regan），神经科学（Horrocks）和超分辨率显微镜技术（Horrocks）方面的专业知识。培训将主要通过在主管和合作者的实验室中实际应用研究技术来实现。将开发的技能包括细胞和分子生物学技术，细胞培养（包括分化诱导多能干细胞），基因编辑，高级显微镜（单分子和超分辨率），数据分析和编码。学生将被鼓励参加培训工作坊，在大学的各种多小组会议上发言，并参加公众参与活动。科学是一项全球性的努力，导师将确保学生有机会参加和出席国际会议。

Fluorescence microscopy allows the dynamic observation of phenomena in living cells. Until recently, however, the wavelike nature of light and its associated diffraction, limited the resolution of optical microscopy to ~200 nm. New 'super-resolution' fluorescence microscopy methods, have been developed to overcome the diffraction limit of 'traditional' microscopy. The enormous impact of super resolution microscopy methods was recognized by its inventors being awarded the Nobel Prize in Chemistry (2014). Super resolution methods can increase the resolution of fluorescence microscopy down to ~5 nm, thus enabling the observation of molecular processes in great detail. Despite these advances in optical methods, labelling the biomolecule of interest remains a challenge, especially within live cells. Direct fusion of the protein of interest to a fluorescent protein is common strategy, because it is genetically encodable, can be used for live cell imaging, and is amenable to super-resolution microscopy (by judicious choice of fluorescent protein). Unfortunately, fluorescent proteins are quite large (>27 kDa) and a direct fusion can perturb the function, assembly, and/or location of the protein of interest.To address these issues, the student will develop a novel super-resolution imaging method based on a labelling strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the biomolecule of interest. Rather than fusing the fluorescent protein itself to the protein of interest, a short peptide tag will be genetically encoded onto it. This tag can then be linked either covalently or non-covalently to a partner protein that is also expressed, in a controlled fashion, within the cell. Such a method has been demonstrated to work well to label proteins within bacteria and yeast1,2. The student will build on this work, adapting the strategy for use in mammalian cells. Most importantly, they will develop this approach using the photoswitchable fluorescent protein, mEoS, which will enable super-resolution images to be generated using photo-activated localization microscopy3. We are particularly interested in applying this novel strategy to visualize amyloid protein formed within human-derived neurons. Training: the supervisors of this project will combine their expertise in protein structure and design (Regan), neuroscience (Horrocks) and super-resolution microscopy techniques (Horrocks). The training will be achieved primarily through practical application of the research techniques within the supervisors' and collaborators' laboratories. Skills that will be developed include cell and molecular biology techniques, cell culture (including differentiated induced pluripotent stem cells), gene editing, advanced microscopy (single-molecule and super-resolution), data analysis and coding. The student will be encouraged to participate in training workshops, to present in various multi-group meetings at the University, and to participate in public engagement activities. Science is a global endeavour, and the supervisors will ensure that the student has the opportunity to attend and present at international conferences.