Salmon Edward D, Shaw Sidney L, Waters Jennifer C, Waterman-Storer Clare M, Maddox Paul S, Yeh Elaine, Bloom Kerry
Department of Biology, University of North Carolina, Chapel Hill, North Carolina, USA.
Methods Cell Biol. 2013;114:179-210. doi: 10.1016/B978-0-12-407761-4.00009-9.
This chapter describes the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope, and a cooled charge-coupled device (CCD) camera. The three main parts of this imaging system are Nikon FXA microscope, Hamamatsu C4880 cooled CCD camera, and MetaMorph digital imaging system. This chapter presents various design criteria for the instrument and describes the major features of the microscope components-the cooled CCD camera and the MetaMorph digital imaging system. The Nikon FXA upright microscope can produce high resolution images for both epifluorescent and transmitted light illumination without switching the objective or moving the specimen. The functional aspects of the microscope set-up can be considered in terms of the imaging optics, the epi-illumination optics, the transillumination optics, the focus control, and the vibration isolation table. This instrument is somewhat specialized for microtubule and mitosis studies, and it is also applicable to a variety of problems in cellular imaging, including tracking proteins fused to the green fluorescent protein in live cells. The instrument is also valuable for correlating the assembly dynamics of individual cytoplasmic microtubules (labeled by conjugating X-rhodamine to tubulin) with the dynamics of membranes of the endoplasmic reticulum (labeled with DiOC6) and the dynamics of the cell cortex (by differential interference contrast) in migrating vertebrate epithelial cells. This imaging system also plays an important role in the analysis of mitotic mutants in the powerful yeast genetic system Saccharomyces cerevisiae.
本章介绍了一种基于宽视场落射荧光和透射光显微镜以及冷却电荷耦合器件（CCD）相机的高分辨率多模式数字成像系统的开发。该成像系统的三个主要部分是尼康FXA显微镜、滨松C4880冷却CCD相机和MetaMorph数字成像系统。本章介绍了该仪器的各种设计标准，并描述了显微镜组件（冷却CCD相机和MetaMorph数字成像系统）的主要特点。尼康FXA直立式显微镜无需更换物镜或移动样本，即可分别针对落射荧光和透射光照明生成高分辨率图像。显微镜设置的功能方面可从成像光学、落射照明光学、透射照明光学、聚焦控制和隔振台等方面进行考虑。该仪器在某种程度上专门用于微管和有丝分裂研究，也适用于细胞成像中的各种问题，包括追踪活细胞中与绿色荧光蛋白融合的蛋白质。该仪器对于关联单个细胞质微管（通过将X-罗丹明与微管蛋白结合进行标记）的组装动力学与内质网（用DiOC6标记）的膜动力学以及迁移的脊椎动物上皮细胞中细胞皮层的动力学（通过微分干涉对比）也很有价值。这种成像系统在强大的酵母遗传系统酿酒酵母中有丝分裂突变体的分析中也发挥着重要作用。
