Expanded dimensionality and high sensitivity cell imaging using designed OMFPs

使用设计的 OMFP 进行扩展维度和高灵敏度细胞成像

• 批准号: 9035801
• 负责人: ROBERT M DICKSON
• 金额: $ 22.07万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9035801
• 关键词: Affect; Amino Acids; Back; Benchmarking; Biology; Biosensor; Cell physiology; Cells; Cellular biology; Characteristics; Color; Complex; Coupled; Detection; Development; Discrimination; Engineering; Environment; Excision; Exclusion; Exhibits; Fluorescence; Fluorescence Microscopy; Frequencies; Green Fluorescent Proteins; Image; Imagery; Imaging Techniques; Information Retrieval; Kinetics; Label; Lasers; Life; Lighting; Location; Measurement; Measures; Methods; Microscope; Mutate; Mutation; Optics; Population; Process; Proteins; Recovery; Relative (related person); Research Personnel; Resolution; Scheme; Scientific Advances and Accomplishments; Signal Transduction; Signaling Protein; Source; Surveys; Time; Tyrosine; base; cellular imaging; chromophore; design; fluorescence imaging; fluorophore; imaging modality; imaging system; improved; interest; optical spectra; physical property; protein protein interaction; public health relevance; red fluorescent protein; response; tool

 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.

 描述(申请人提供)：生物学已经建立了一系列高度复杂和动态的网络，这些网络几乎管理着所有的细胞过程。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的位置、相互作用和动力学的首选标记。FPS提供的便利往往超过了与自动荧光背景、中等亮度和有限的可分辨颜色相关的挑战。我们提出的研究将开发一类重要的新发射体-光调制荧光蛋白(OMFP)。通过利用与每个OMFP相关的唯一时间尺度来建立荧光限制的暗态群体，我们将能够1)将所需信号从所有(不可调制的)背景中分离出来，以及2)从多个否则在光谱上无法区分的发射体中区分信号。通过有针对性的突变来稳定各种发色团状态，我们将调整每个OMFP的调制深度与调制频谱。由此产生的独特的调制频率响应将被用于扩展荧光成像的维度，以同时区分相同光谱区域内至少3倍以上的荧光团，同时排除所有不可调制的自动荧光背景。将在每种颜色中产生多个OMFP-从蓝色到红色，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动单元中被空间分辨。调制光谱的这种光谱分解，加上通过光学解调去除背景，将通常适用于同时跟踪单个光谱区域内的多个发射体。将对灵敏度进行基准测试，以证明所设计的材料和方法的优势。我们将使用这些方法在单个单元中同时成像多达6个发射器，并使用商用共聚焦显微镜推动使用光学调制的方法来显著提高灵敏度。这项工作将提供一套通用的工具、方法和总体框架，用于在活细胞的复杂环境中调查和测量多个重叠的蛋白质位置。