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（由适用提供）：生物学已经建立了一系列高度复杂和动态的网络，这些网络几乎控制了所有细胞过程。对于理解这些过程至关重要，荧光蛋白（FPS）是表征活细胞中蛋白质位置，相互作用和动力学的选择标签。 FPS提供的便利通常超过与自动荧光背景，中等亮度和有限区分颜色相关的挑战。我们提出的研究将开发一类新的发射器 - 光学调制荧光蛋白（OMFP）。通过利用与每个OMFP相关的独特时间尺度来堆积荧光限制的黑暗状态人群，我们将能够1）1）与所有人（不可变化）背景和2）不同的信号与多个频谱不可区分的发射器不同。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。所得的独特调制频率响应将被用来扩大荧光成像的维度，以简单地区分同一光谱区域内的荧光团至少3倍，同时拒绝所有无法建模的自动荧光背景。每种颜色将产生多个OMFP-蓝色至红色，并且在给定的颜色中，至少三个光谱相似的EMITTR信号将根据其独特的调制频率响应在实时单元中在空间上解析。调制光谱的这种频谱不混合，再加上通过光学解调的背景去除，通常适用于单个光谱区域内的多个EMITTR。将进行灵敏度的基准测试，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单元中轻松映像多达6个发射器，并推动使用商业共聚焦显微镜的光学调制，以极大地提高灵敏度。这项工作将提供一组一般的工具，方法和整体框架，用于测量和测量活细胞复杂环境中多个重叠蛋白质位置。