Expanded dimensionality and high sensitivity cell imaging using designed OMFPs

 描述（申请人提供）：生物学已经建立了一套庞大的高度复杂和动态的网络，几乎所有的细胞过程。对于理解这些过程至关重要，荧光蛋白（FP）是表征活细胞中蛋白质位置，相互作用和动力学的首选标签。FP提供的便利通常超过与自动荧光背景、中等亮度和有限的可区分颜色相关的挑战。我们提出的研究将开发一种重要的新型发光体-光调制荧光蛋白（OMFP）。通过利用与每个0 MFP相关联的独特时间尺度来建立荧光限制暗态群体，我们将能够1）将期望的信号与所有（不可调制的）背景分离，以及2）区分来自多个否则光谱上不可区分的发射体的信号。通过稳定各种发色团状态的靶向突变，我们将调整每个OMFP的调制深度与调制频谱。所得到的独特调制频率响应将用于扩展荧光成像的维度，以同时区分相同光谱区域内的至少3倍以上的荧光团，同时拒绝所有不可调制的自动荧光背景。多个OMFP将以每种颜色产生-蓝色到红色，并且在给定的颜色内，至少三个光谱相似的发射信号将基于其独特的调制频率响应在活细胞中空间分辨。调制光谱的这种光谱解混，与通过光学解调的背景去除相结合，通常将适用于同时跟踪单个光谱区域内的多个发射器。将进行灵敏度基准测试，以证明设计材料和方法的优势。我们将采用这些方法在单个细胞中同时成像多达6个发射器，并推动采用光学调制的方法，以使用商业共聚焦显微镜大幅提高灵敏度。这项工作将提供一套通用的工具，方法和整体框架，用于调查和测量活细胞复杂环境中的多个重叠蛋白质位置。

 DESCRIPTION (provided by applicant): Biology has built up a vast set of highly complex and dynamic networks that govern nearly all cellular processes. Crucial to understanding these processes, fluorescent proteins (FPs) are the label of choice in characterizing protein location, interactions, and dynamics in live cells. The convenience afforded by FPs often outweighs challenges associated with auto fluorescent background, moderate brightness, and limited distinguishable colors. Our proposed studies will develop an important new class of emitters - optically modulated fluorescent proteins (OMFPs). By taking advantage of the unique timescale associated with each OMFP for the buildup of a fluorescence-limiting dark state population, we will be able to 1) separate desired signals from all (unmodulatable) background and 2) distinguish signals from multiple, otherwise spectrally indistinguishable emitters. Through targeted mutations that stabilize various chromophore states, we will tune the modulation depth vs. modulation frequency spectrum of each OMFP. The resulting unique modulation frequency responses will be utilized to expand the dimensionality of fluorescence imaging to simultaneously distinguish at least 3-fold more fluorophores within the same spectral regions, all while rejecting all unmodulatable auto fluorescent background. Multiple OMFPs will be produced in each color - blue through red, and within a given color, at least three spectrally similar emittr signals will be spatially resolved in live cells based on their unique modulation frequency responses. This spectral unmixing of modulation spectra, coupled with background removal through optical demodulation, will be generally applicable to simultaneously follow multiple emitters within a single spectral region. Benchmarking of sensitivities will be performed to demonstrate advantages of the designed materials and methods. We will employ these methods to simultaneously image up to 6 emitters in a single cell, and push methods to employ optical modulation for drastically improved sensitivity using commercial confocal microscopes. This effort will provide a general set of tools, methods, and overall framework for surveying and measuring multiple overlapping protein locations within the complex environment of live cells.