A Confocal Laser Scanning Microscope for Cellular and Molecular Imaging

该提案要求为一种共聚焦激光扫描显微镜系统提供资金，该系统基于具有差干涉和荧光能力的高分辨率成像的红外校正蔡司Axiovert显微镜。共聚焦显微镜将有一个氪/氩激光器，三个光电倍增管（PMT）和一个机动透射光PMT。该显微镜将用于成像各种细胞和分子结构的动态和静态标本在高分辨率和三维细节。需要不同的记录设备。光盘和CD-ROM单元将以数字方式存储图像，而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盘记录仪记录。这项提议来自19位科学家，他们分别来自北卡罗莱纳州立大学的9个不同系和3个学院，以及维克森林大学的一个系。据我们所知，没有其他的共聚焦激光扫描显微镜用于精确的高分辨率成像，在两个校园可用。7位项目负责人中的大多数是植物生物学家，他们使用涉及遗传、分子、生化和细胞生物学技术的组合方法来研究基本的细胞、发育和分子过程，并且都非常需要共聚焦显微镜。这些项目包括研究细胞骨架和细胞信号传导（特别是钙离子）之间的关系，因为它影响植物发育（Boss, Allen, Davies, Muday），玉米储存蛋白的靶向（Boston），病毒发病和运输（Robertson和Lommel），线虫发病和根特异性基因表达（Conkling），以及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，最初将是少量使用者，包括以下人员：动物学组的三名成员将研究上皮细胞（Black）、神经元和神经胶质细胞（Grossfeld）以及鱼的脑垂体（Borski）中的细胞信号传导的各个方面。Mahaffey（遗传学）将研究果蝇幼虫的片段，Weissinger将研究转基因植物中的真菌发病机制。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。穆迪将研究植物中的生长素运输。最后在植物学，伯克霍尔德将研究鞭毛藻的生命周期。荧光分子或荧光结构的精确定位和解析对于许多此类研究至关重要。共聚焦显微镜在高分辨率下制作非常薄的光学切片的独特能力在过去十年中开辟了细胞生物学的新领域，这组科学家已经意识到通过应用共聚焦成像和图像分析和定量的分子染色的新方法可以大大增强他们的研究工作。北卡罗莱纳州立大学和植物系已经做出了真正的承诺，以加强细胞生物学和细胞成像。这种对成像的兴趣部分是由于生物技术和分子生物学方向的教师需要将先进的成像技术纳入他们的工作中。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。植物学和动物学最近聘请了几位细胞生物学家。一个细胞，分子和发育生物学项目正在开发中，将包括来自许多不同部门的教师。Nina Allen将领导细胞和分子成像中心（CMIC），该中心将设在植物学系，该区域正在为Allen和Davies翻新。用NCSU提供给Allen的安装资金购买了几台高分辨率视频显微镜。温迪·博斯已经获得了资金，购买了一个冷却ccd成像系统，该系统将被安置在CMIC。这些显微镜与共聚焦显微镜相结合，将使中心的成像能力具有很大的灵活性，并将大大加强大学许多领域的研究。

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.