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野荧光显微镜，用于 3D 图像采集、反卷积以及活细胞延时和光漂白后荧光恢复 (FRAP)。通过使用 10 MHz Cascade II EMCCD 相机与反卷积软件相结合，可以实现 3D 延时的高灵敏度和快速数据采集。该仪器将为不同用户群体提供灵活的活体和固定哺乳动物和酵母细胞以及组织切片成像。富勒实验室正在研究酵母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形成和消耗的动态，以及使用功能性荧光蛋白与内源蛋白质的融合作为探针，测量运输蛋白质与细胞器的关联。恩格尔克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复杂且空间受控的基因定位，并且这种定位是动态响应代谢和发育信号的。目前正在研究 RNA 聚合酶 III 启动子聚类的机制，作为基因组压缩和组织的一种手段。柯林斯实验室专注于人类免疫缺陷病毒 (HIV-1) 如何逃避免疫系统以建立持续感染。 Collins 和他的同事已经证明，实现这一目标的一种方法是通过 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 HIV-1 Nef 蛋白对原代 T 细胞和 T 细胞系中 MHC-I、其他膜蛋白以及外壳蛋白和衔接蛋白的影响。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 细胞激活过程中细胞外和细胞内氧化还原电位的重塑及其调节性 T 细胞的调节。他们使用荧光传感器来跟踪外表面蛋白质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。该仪器（和感兴趣的领域）的小用户将包括 Carol Fierke 教授（锌代谢的划分）、Tom Kerppola 教授（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）。