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（由申请人提供）：生物学已经建立了大量高度复杂和动态的网络，它们控制着几乎所有的细胞过程，荧光蛋白（FP）是表征蛋白质位置、相互作用、 FP 提供的便利性通常超过了与自动荧光背景、中等亮度和有限的可区分颜色相关的挑战。 (OMFP) 通过利用与每个 OMFP 相关的独特时间尺度来构建荧光限制暗态群体，我们将能够 1) 从所有（不可调节的）背景中分离出所需信号，2) 从多个背景中区分出信号。 ，否则在光谱上无法区分的发射器。通过稳定各种发色团状态的目标突变，我们将调整每个 OMFP 的调制深度与调制频谱，从而利用所得的独特调制频率响应。扩大荧光成像的维度，以同时区分相同光谱区域内至少 3 倍的荧光团，同时拒绝所有不可调节的自动荧光背景，每种颜色（蓝色到红色）以及给定颜色内都会产生多个 OMFP。 ，至少三个光谱相似的发射器信号将根据其独特的调制频率响应在活细胞中进行空间解析，加上通过光学解调的背景去除。通常适用于同时跟踪单个光谱区域内的多个发射器，我们将进行灵敏度基准测试，以证明所设计的材料和方法的优点，我们将采用这些方法在单个单元中同时对多达 6 个发射器进行成像。使用商业共聚焦显微镜采用光学调制来显着提高灵敏度这项工作将为调查和测量活细胞复杂环境中的多个重叠蛋白质位置提供一套通用的工具、方法和总体框架。