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(申请人提供)：这是一个多用户应用程序购买倒置，宽视场荧光显微镜具有高灵敏度和分辨率的3D图像采集，去卷积，活细胞时间推移和荧光恢复后光漂白(FRAP)。利用10 MHz Cascade II EMCCD相机结合反褶积软件，可以实现高灵敏度和快速的三维时间推移数据采集。该仪器将为不同的用户群体提供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灵活性。富勒实验室正在研究酵母中高尔基体晚期和内体隔间之间运输的分子基础。该仪器将被用来测量运输囊泡的形成和消耗的动力学，以及运输蛋白与细胞器的联系，使用功能性荧光蛋白与内源蛋白的融合作为探针。恩格尔克实验室正在研究萌芽酵母、小鼠细胞和人类细胞中核基因组的三维结构。细胞核中的信息检索涉及复杂的和空间控制的基因定位，这种定位是对代谢和发育信号的动态响应。目前正在研究将RNA聚合酶III启动子聚集作为基因组浓缩和组织的一种手段的机制。柯林斯实验室专注于人类免疫缺陷病毒(HIV-1)如何在建立持续感染的过程中逃避免疫系统。Collins及其同事已经证明，实现这一目标的一种方法是通过HIV-1Nef蛋白的活性，它通过改变MHC-I的细胞内运输来扰乱抗原递送。3D和4D成像将被用于绘制MHC-I、其他膜蛋白以及原代T细胞和T细胞系中的外壳和接头蛋白的影响图。实验将评估nef相互作用部位的突变和细胞运输因子的敲除的影响。Banerjee实验室正在研究效应器T细胞激活过程中细胞外和细胞内氧化还原电位的重塑以及调节性T细胞对其调节。他们使用荧光传感器跟踪表面蛋白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。该仪器的次要使用者(和感兴趣的领域)将包括Carol Fierke教授(锌代谢的区隔)，Tom Kerppola教授(细胞泛素化途径，双分子荧光互补)，Stephen Ragsdale教授(血红素加氧酶-2相互作用)，Anne Vojtek教授(调节神经元形态发生的信号机制)，Aaron Goldstrohm教授(RNA稳定性和降解)。