Nikon TiE Motorized Microscope System For 3D Image Acquisition, Deconvolution, Li

Nikon TiE 电动显微镜系统，用于 3D 图像采集、反卷积、Li

• 批准号: 7794471
• 负责人: ROBERTA S. FULLER
• 金额: $ 31.67万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-10 至 2011-09-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7794471
• 关键词: Adaptor Signaling Protein; Antigen Presentation; Area; Cell Line; Cell Nucleus; Cells; Chimeric Proteins; Complex; Computer software; Consumption; Development; Fluorescence; Fluorescence Recovery After Photobleaching; Gene Order; Genome; Glutathione; Glutathione Disulfide; Golgi Apparatus; HIV; HIV-1; Human; Image; Immune system; Infection; Information Retrieval; Laboratories; Life; Maps; Measures; Membrane Proteins; Metabolic; Metabolism; Microscope; Minor; Molecular; Morphogenesis; Mus; Mutation; Neurons; Nuclear; Organelles; Oxidation-Reduction; Pathway interactions; Physical condensation; Positioning Attribute; Proteins; RNA Polymerase III; RNA Stability; Regulatory T-Lymphocyte; Resolution; Saccharomyces cerevisiae; Saccharomycetales; Signal Transduction; Site; Slice; System; T-Cell Activation; T-Lymphocyte; Three-Dimensional Image; Time; Tissues; Transport Vesicles; Ubiquitination; Yeasts; Zinc; base; data acquisition; flexibility; fluorescence microscope; heme oxygenase-2; instrument; interest; nef Protein; professor; promoter; protein transport; research study; response; sensor; trafficking

DESCRIPTION (provided by applicant): This is a multi-user application to purchase an inverted, widefield fluorescence microscope with high sensitivity and resolution for 3D image acquisition, deconvolution, and live cell time lapse and fluorescence recovery after photobleaching (FRAP). High sensitivity and rapid data acquisition for 3D time lapse will be achieved by using a 10 MHz Cascade II EMCCD camera combined with deconvolution software. The instrument will provide flexibility for use in imaging live and fixed mammalian and yeast cells as well as tissue slices for a diverse group of users. The Fuller laboratory is studying the molecular basis of trafficking between late Golgi and endosomal compartments in yeast. This instrument will be used to measure the dynamics of transport vesicle formation and consumption and of the association of trafficking proteins with organelles using, as probes, functional fluorescent-protein fusions to endogenous proteins. The Engelke lab is examining the three-dimensional organization of nuclear genomes in the budding yeast, Saccharomyces cerevisiae, and in murine and human cells. Information retrieval in nuclei involves complex and spatially controlled positioning of genes, and this positioning is dynamic in response to metabolic and developmental signals. The mechanism is being investigated for clustering of RNA polymerase III promoters as a means of genome condensation and organization. The Collins lab is focused on the how the human immunodeficiency virus (HIV-1) evades the immune system in establishing a persistent infection. Collins and coworkers have shown that one way this is accomplished is via the activity of the HIV- 1 Nef protein, which disrupts antigen presentation by altering the intracellular trafficking of MHC- I. 3D and 4D imaging will be used to map the effects of on MHC-I, other membrane proteins and coat and adaptor proteins in primary T-cells and T-cell lines. Experiments will evaluate effects of mutations in nef interaction sites and knockdowns of cellular transport factors. The Banerjee lab is studying remodeling of the extra- and intra-cellular redox potential during effector T cell activation and its modulation by regulatory T cells. They use fluorescent sensors to track changes in the redox status of exofacial proteins and for the direct readout of the intracellular glutathione: glutathione disulfide redox potential. Minor users of the instrument (and areas of interest) will include Professors Carol Fierke (compartmentation of zinc metabolism), Tom Kerppola (cellular ubiquitination pathways, bimolecular fluorescence complementation), Stephen Ragsdale (heme oxygenase-2 interactions), Anne Vojtek (signaling mechanisms regulating neuronal morphogenesis), Aaron Goldstrohm (RNA stability and degradation).

描述（由申请人提供）：这是一个多用户应用程序，用于购买具有高灵敏度和分辨率的倒置宽视场荧光显微镜，用于3D图像采集、去卷积、活细胞延时和光漂白后荧光恢复（FRAP）。使用10 MHz Cascade II EMCCD照相机结合反卷积软件，将实现三维时间推移的高灵敏度和快速数据采集。该仪器将提供灵活性，用于成像活的和固定的哺乳动物和酵母细胞，以及组织切片的不同群体的用户。富勒实验室正在研究酵母中晚期高尔基体和内体区室之间运输的分子基础。该仪器将用于测量运输囊泡的形成和消耗的动力学，以及使用作为探针的功能性荧光蛋白与内源性蛋白质的融合物来测量运输蛋白与细胞器的关联。Engelke实验室正在研究芽殖酵母、酿酒酵母以及小鼠和人类细胞中核基因组的三维组织。细胞核中的信息检索涉及基因的复杂和空间控制的定位，并且这种定位是动态的，以响应代谢和发育信号。正在研究RNA聚合酶III启动子聚集作为基因组浓缩和组织手段的机制。柯林斯实验室的重点是人类免疫缺陷病毒（HIV-1）如何逃避免疫系统建立一个持续的感染。柯林斯和同事已经表明，实现这一点的一种方式是通过HIV- 1 Nef蛋白的活性，该蛋白通过改变MHC-I的细胞内运输来破坏抗原呈递。3D和4D成像将用于绘制对原代T细胞和T细胞系中MHC-I、其他膜蛋白以及外壳和衔接蛋白的影响。实验将评估nef相互作用位点突变和细胞转运因子敲低的影响。Banerjee实验室正在研究效应T细胞活化过程中细胞外和细胞内氧化还原电位的重塑及其受调节性T细胞的调节。他们使用荧光传感器来跟踪外表面蛋白的氧化还原状态的变化，并直接读出细胞内谷胱甘肽：谷胱甘肽二硫化物氧化还原电位。仪器的小用户（和感兴趣的领域）将包括Carol Fierke教授（锌代谢的区室化），Tom Kerppola（细胞泛素化途径，双分子荧光互补），Stephen Ragsdale（血红素加氧酶-2相互作用），Anne Vojtek（调节神经元形态发生的信号机制），Aaron Goldstrohm（RNA稳定性和降解）。