A Confocal Laser Scanning Microscope for Cellular and Molecular Imaging

该提案要求提供资金，用于基于红外校正的蔡司Axiovert显微镜的共聚焦激光扫描显微镜系统，该显微镜具有差分干涉和荧光能力，可进行高分辨率成像。共聚焦显微镜将有一个氪/氩激光器，三个光电倍增管（PMT）和一个电动透射光PMT。该显微镜将用于以高分辨率和三维细节对动态和静态标本中的各种细胞和分子结构进行成像。要求使用不同的记录设备。一个光盘和一个CD-ROM单元将以数字方式存储图像，一个硬拷贝彩色打印机将允许快速检索图像。动态结构将由松下光学存储盘记录器记录 这项提案来自北卡罗来纳州州立大学的9个不同系和3个学院以及维克森林大学的一个系的19名科学家。据我们所知，没有其他共焦激光扫描显微镜的精确高分辨率成像提供在任何校园。七个项目的领导者大多是植物生物学家，他们使用涉及遗传、分子、生物化学和细胞生物学技术的组合方法研究基本的细胞、发育和分子过程，并且都非常真实的需要共聚焦显微镜。这些项目包括研究细胞骨架和细胞信号之间的关系（特别是钙离子），因为它影响植物的发育（Boss，艾伦，Davies，Muday），在病毒发病机理和运输的玉米（Boston）中靶向储存蛋白（Robertson和洛梅尔），线虫发病机理和根特异性基因表达（Conkling），和铁氧还蛋白定位以及植物细胞中的核支架附着区（Thompson）。此外，Peretti将研究生物膜中的质粒转移。另一组研究人员最初将是少数用户，包括以下人员：动物学的三名成员将研究上皮细胞（布莱克）、神经元和神经胶质（格罗斯菲尔德）以及鱼类脑垂体（博尔斯基）中细胞信号的各个方面。Mahaffey（遗传学）将研究果蝇幼虫的分节，Weissinger将研究转基因植物中的真菌致病机理。Foegeding（食品科学）将研究生物聚合物凝胶的生物流变特性，Laster（微生物学）将研究肿瘤诱导的细胞凋亡。穆迪将研究植物中生长素的运输。最后在植物学中，伯克霍尔德将研究甲藻的生命周期。对于许多这些研究来说，荧光分子或结构被精确定位和解析是必不可少的。在过去的十年里，共聚焦显微镜以高分辨率制作非常薄的光学切片的独特能力开辟了细胞生物学的新前沿，这组科学家已经意识到通过应用分子染色与共聚焦成像和图像分析和定量的新方法来大大提高他们的研究成果的机会。 北卡罗来纳州州立大学和植物学系做出了非常真实的承诺，加强细胞生物学和细胞成像。这种对成像的兴趣部分是由生物技术和分子生物学导向的教师需要将先进的成像技术纳入他们的工作。例如，现在可以使用分子生物学技术在植物中表达转基因荧光探针如绿色荧光蛋白。最近，植物学和动物学聘请了几位细胞生物学家。一个细胞，分子和发育生物学计划正在开发中，将包括来自许多不同部门的教师。尼娜艾伦将指导细胞和分子成像中心（CMIC）将被安置在植物系在该地区正在装修的艾伦和戴维斯。 几个高分辨率的视频显微镜已购买的设置资金由NCSU提供给艾伦。温迪·博斯已经获得了购买一个冷却CCD成像系统的资金，该系统将安装在CMIC中。 这些显微镜与共聚焦显微镜相结合，将允许在中心提供成像能力的巨大灵活性，并应大大加强在大学的许多研究领域。

This proposal requests funds for a confocal laser scanning microscope system based on an infmity corrected Zeiss Axiovert microscope with both differential interference and fluorescence capabilities for high resolution imaging. The confocal microscope will have a Krypton/Argon laser, three photomultiplier tubes (PMT) and a motorized transmitted light PMT. This microscope will be used to image a variety of cellular and molecular structures in both dynamic and still specimens at high resolution and in 3-dimensional detail. Different recording devices are requested. An optical disc and a CD-ROM unit will store images digitally and a hard copy color printer will allow quick retrieval of images. Dynamic structures will be recorded by a Panasonic Optical Memory Disc Recorder This proposal comes from 19 scientists from 9 different Departments and three Colleges at North Carolina State University and one Department at Wake Forest University. To our knowledge there is no other confocal laser scanning microscope for precise high resolution imaging available on either campus. Most of the seven project leaders are plant biologists investigating basic cellular, developmental and molecular processes using a combinatorial approach involving genetic, molecular, biochemical, and cell biological techniques and all have a very real need of confocal microscopy. The projects include investigations of the relationships between cytoskeleton and cell signaling (especially Calcium ions) as it influences plant development (Boss, Allen, Davies, Muday), of the targeting of storage proteins in maize (Boston) of viral pathogenesis and transport (Robertson and Lommel), of nematode pathogenesis and root specific gene expression (Conkling), and of ferredoxin localization as well as nuclear scaffold attachment regions in plant cells (Thompson). In addition, Peretti will study plasmid transfer in biofilms. Another group of investigators, who initially will be minor users, include the following: Thr ee members of Zoology will study various aspects of cell signaling in epithelial cells (Black), neurons and glia (Grossfeld) and from pituitary glands of fish (Borski). Mahaffey (Genetics) will study segmentation in Drosophila larvae and Weissinger will investigate fungal pathogenesis in transgenic plants. Foegeding (Food Science) will study biorheological properties of biopolymer gels and Laster (Microbiology) will investigate tumor induced apoptosis. Muday will study auxin transport in plants. Finally in Botany, Burkholder will study dinoflagellate life cycles. It is essential for many of these studies that fluorescent molecules or structures be precisely localized and resolved. The unique ability of the confocal microscope to make very thin optical sections at high resolution has opened new frontiers in cell biology in the last ten years, and this group of scientists has realized the opportunities available to greatly enhance their research efforts by applying the newer methods of molecular staining with confocal imaging and image analysis and quantification. North Carolina State University and the Department of Botany have made a very real commitment to strengthen cell biology and cellular imaging. This interest in imaging was driven in part by a need of the biotechnology and molecular biology oriented faculty to incorporate advanced imaging in their work. For example, it is now possible to use molecular biology techniques to express transgenically fluorescent probes such as Green Fluorescent Protein in plants. Several cell biologists have recently been hired in Botany and Zoology. A Cellular, Molecular and Developmental Biology Program is in development and will include faculty from many diverse Departments. Nina Allen will direct the Cellular and Molecular Imaging Center (CMIC) to be housed in the Botany Department in the area being renovated for Allen and Davies. Several high resolution video microscopes have been purchased with set-up money provided by NCSU to Allen. Wendy Boss has obtained funds to purchase a cooled-CCD imaging system that will be housed in CMIC. These microscopes in combination with the confocal microscope will allow great flexibility in imaging capabilities available in the Center and should greatly strengthen many areas of research at the University.