Upgrade of Confocal Microscope for Use in Developmental and Cell Biological Studies

申请的资金将用于升级Bio-Rad MRC-600共焦显微镜。将购买的物品包括：一个光纤安装的氪/氩激光器，改进的光电倍增管，一个60X 1.2na的浸水镜头，以及一台用于图像处理的计算机。这种新的激光器将极大地改善双标记免疫荧光实验中的颜色分离，并允许进行三标记实验。光纤支架将减少扫描头的振动，并在高倍率下提高分辨率。新的光电倍增管将允许成像较暗的物体，水浸透镜将提高厚样品的图像质量。通过让第二台计算机进行离线处理，一名研究人员可以在另一名研究人员处理数据的同时收集数据。该设备将用于细胞生物学和发育实验。细胞生物学实验的中心是分子在细胞中的运输。将进行研究，以了解果蝇无翼分泌蛋白是如何被细胞摄取的，以及跨细胞作用在无翼蛋白梯度形成中的潜在作用。这些实验应该有助于更好地理解发育中的生物体是如何产生模式的。在其他工作中，将研究RAB7蛋白在内吞途径中的作用。这项工作的目标是了解分子是如何针对各种细胞器的。细胞生物学研究的第三个主要领域是使用阳离子脂类将分子输送到细胞内。这项技术将被用于研究各种细胞过程。所有这些研究都需要共同本地化实验，其中最大限度地实现颜色分离是至关重要的。此外，跟踪分子的亚细胞分布的能力需要尽可能好的分辨率和最高的灵敏度。发育研究主要集中在果蝇上。除了上面描述的Wingle ss实验，正在进行关于腿部模式的转录调节以及细胞谱系在中枢神经系统模式中的作用的研究。这两项研究都需要能够进行三重标记实验，其中三个分子可以同时跟踪。腿部模式实验的目标是了解位置信息的梯度是如何解释的，并导致对单个细胞命运的指定。神经元谱系实验将检验谱系和位置信息对单个神经元最终表型的相对贡献。氪/氩激光对于三重标记实验是必不可少的，浸水透镜将允许神经元及其轴突投射在整个胚胎过程中得到跟踪。

The requested funds will be used to upgrade a Bio-Rad MRC-600 confocal microscope. The items to be purchased are: a Krypton/Argon laser with fiber optic mount, improved photomultiplier tubes, a 60X 1.2 NA water immersion lens, and a computer for image processing. The new laser will dramatically improve the color separation in double label immunofluorescence experiments and allow triple label experiments to be performed. The fiber optic mount will decrease vibrations in the scan head and improve resolution at high magnification. The new photomultiplier tubes will allow the imaging of fainter objects and the water immersion lens will improve the quality of images from thick specimens. By having a second computer for off line processing, one researcher can be collecting data while a second is processing data. The equipment will be used in both cell biological and developmental experiments. The cell biological experiments center on the trafficking of molecules through the cell. Studies will be performed to understand how the Drosophila Wingless secreted protein is taken up by cells and the potential role of transcytosis in the formation of a gradient of Wingless protein. These experiments should lead to a better understanding of how pattern is generated in developing organisms. In other work the role of the Rab 7 protein in the endocytic pathway will be examined. The goal of this work is to understand how molecules are targeted to various organelles. The third main area of cell biological study is the use of cationic lipids for delivering molecules into cells. This technology will be used to study a variety of cellular processes. All these studies require co-localization experiments in which it is critical to maximize color separation. In addition, the ability to follow the subcellular distribution of molecules requires the best possible resolution and highest sensitivity. The Developmental studies are focused on Drosophila. In addition to the Wingle ss experiments described above, research is being conducted on the transcriptional regulation of leg patterning and the role of cell lineage in the patterning of the central nervous system. Both of these studies require an ability to carry out triple label experiments in which three molecules can be followed simultaneously. The goal of the leg patterning experiments is to understand how a gradient of positional information is interpreted and leads to the specification of individual cell fates. The neuronal lineage experiments will examine the relative contribution of lineage and positional information to the final phenotype of individual neurons. The Krypton/Argon laser is essential for the triple label experiments and the water immersion lens will allow neurons and their axonal projections to be followed throughout the embryo.