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.