Fluorescent proteins for superresolution imaging

描述（由申请人提供）：拟议研究的目标是通过开发改进的荧光蛋白变体来扩展随机单分子超分辨率（SSMS）显微镜的能力和性能。该项目的主要目标是使SSMS能够同时在活细胞中对三种或更多种蛋白质进行成像，从而在生物相关背景下可视化多蛋白质复合物的动力学，单分子定位精度为20 nm或更低。目前，活细胞SSMS成像主要限于单色和双色标记，这是因为缺乏在适当波长下表现良好的荧光蛋白变体。合适的荧光蛋白的短缺 标记来自于依赖于来自非常有限数量的物种的荧光蛋白作为工程化改进变体的起始材料。该项目将利用具有新光学特性的野生型荧光蛋白，从广泛的物种中克隆，包括通过国际合作提供的几种大堡礁珊瑚。这种广泛的新型起始材料的使用将最大限度地提高单体荧光蛋白成功工程的可能性，并且与当前技术相比性能显着改善。具体重点将放在哺乳动物细胞中的功能筛选，而不是专门在细菌中定向进化，这是过去大多数荧光蛋白工程工作的标准方法。在哺乳动物细胞中扩大使用基于图像的筛选将有助于选择在该技术的主要最终用户应用中表现最佳的变体。该项目的最终产品将是三大类单体荧光蛋白变体的成套产品：（1）标准品橙子、红色和远红色波长类别的（非光活性）荧光蛋白，其显示出改善的亮度、光稳定性、作为融合标签的性能和作为SSMS成像标签的性能;（2）在整个可见光谱上显示出与标准荧光蛋白相当的性能的可光活化和光致变色荧光蛋白，而且还包括暗态和激活态之间异常高的对比度;（3）光转换蛋白，专注于开发新的波长类别，包括红-黄、蓝-红和红-远-红，其性能目标与其他两个类别类似。对于这些类别中的每一个，目标将是开发高性能的变体，允许在活细胞中同时成像至少三种不同的蛋白质种类，如通过使用SSMS显微镜的几种生物相关测定所测量的。在这个项目的过程中创建的荧光蛋白变体也将非常有用，作为一般的荧光标签，并预计 是迄今为止最亮、性能最高的荧光蛋白之一。

DESCRIPTION (provided by applicant): The goal of the proposed research is to expand the capabilities and performance of stochastic single- molecule superresolution (SSMS) microscopy through the development of improved fluorescent protein variants. A primary objective of this project will be to enable SSMS imaging in living cells of three or more proteins simultaneously, allowing visualization of multi-protein complex dynamics in a biologically relevant context with single-molecule localization precision of 20 nm or less. Currently, live-cell SSMS imaging is largely limited to one- and two-color labeling, due to the lack of well-performing fluorescent protein variants at appropriate wavelengths. Much of this shortfall in suitable fluorescent protein labels has come from a reliance on fluorescent proteins from a very limited number of species as starting materials for engineering improved variants. This project will take advantage of wild-type fluorescent proteins with novel optical properties to be cloned from a broad range of species, including several Great Barrier Reef corals made available through an international collaboration. The use of this wide array of novel starting materials will maximize the odds of successful engineering of monomeric fluorescent proteins with markedly improved performance over current technology. Specific emphasis will be placed on functional screening in mammalian cells, rather than directed evolution exclusively in bacteria, as has been the standard approach to most past fluorescent protein engineering efforts. This expanded use of image-based screening in mammalian cells will facilitate the selection of variants that perform optimally in th primary end-user applications of this technology. The end products of this project will be complete sets of monomeric fluorescent protein variants in three classes: (1) standard (non-photoactive) fluorescent proteins in the orange, red, and far-red wavelength classes that display improved brightness, photostability, performance as fusion tags, and performance as SSMS imaging tags; (2) photoactivatable and photochromic fluorescent proteins across the entire visual spectrum that display equivalent performance to standard fluorescent proteins, but also include exceptionally high contrast between dark and activated states; (3) photoconvertible proteins, focused on the development of novel wavelength classes including red-to-yellow, blue-to-red, and red-to-far-red, with performance goals similar to the other two classes. For each of these classes, the goal will be development of high-performing variants that allow imaging of at least three distinct protein species simultaneously in living cells, as measured by several biologically relevant assays using SSMS microscopy. The fluorescent protein variants created in the course of this project will also be highly useful as general fluorescent tags, and are expected to be among the brightest and highest- performing fluorescent proteins yet developed.