Multimerized GFP probe for live cell imaging

用于活细胞成像的多聚化 GFP 探针

• 批准号: 9275525
• 负责人: Bo Huang
• 金额: $ 19.81万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9275525
• 关键词: Affect; Animals; Biomedical Research; CRISPR/Cas technology; Cells; Chromosome Structures; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Color; Complement; DNA Sequence; Detection; Development; Event; FarGo; Fluorescence; Fluorescence Microscopy; Genome; Genomics; Image; Individual; Interphase Chromosome; Label; Methods; Microscopy; Mitosis; Mitotic Chromosome; Monitor; Nature; Organizational Change; Photobleaching; Phototoxicity; Process; Protein Engineering; Proteins; Recruitment Activity; Regulation; Reporter; Research; Resolution; Scheme; Signal Transduction; System; Technology; Three-Dimensional Imaging; Time; base; fluorophore; imaging modality; live cell imaging; mutant; single molecule; success; temporal measurement; tool

Project Summary Fluorescence signal level, photobleaching and phototoxicity are major problems encountered ubiquitously in live fluorescence microscopy applications. Because of limited of fluorophore signals, long term monitoring of live cells presents a major challenge to existing fluorescent microscopy technologies. Not only does low signal level affect detection precision over the auto-fluorescence background, the associated photobleaching and phototoxicity also restrict the temporal resolution and observation duration. Inspired by the recent development of the SunTag, we propose to develop a protein labeling scheme using split-fluorescent proteins. By arranged them into tandem arrays and demonstrated proportional signal amplification. This signal amplification can not only solve the signal strength challenge, but also reduce photobleaching / phototoxicity by correspondingly lowering the excitation intensity. Specifically, we plan to develop split-FP tags for multicolor imaging and apply it to live cell imaging of chromosome organization.

项目概要 荧光信号水平、光漂白和光毒性是普遍遇到的主要问题 活体荧光显微镜应用。由于荧光团信号有限，需要长期监测 活细胞对现有荧光显微镜技术提出了重大挑战。不仅信号低 水平影响自发荧光背景的检测精度，相关的光漂白和 光毒性还限制了时间分辨率和观察持续时间。受到最近发展的启发 SunTag 的研究人员建议开发一种使用分裂荧光蛋白的蛋白质标记方案。按安排 将它们组合成串联阵列并展示比例信号放大。这种信号放大不能 不仅解决了信号强度的挑战，还相应地减少了光漂白/光毒性 降低激发强度。具体来说，我们计划开发用于多色成像的 split-FP 标签并应用 它可用于染色体组织的活细胞成像。