A Light Microscope Equipped with Epifluorescence and DIC Optics and an Imaging System

我们建议获得一台奥林巴斯BX-50显微镜，配备荧光、相位和DIC光学元件，以及能够采集和分析高分辨率黑白图像和低分辨率视频速率彩色图像的成像系统。具体地说，我们提出了一种超晶形成像系统，该系统由普林斯顿仪器公司的Pentamax CCD摄像机和DAGE MTI三芯片彩色CCD摄像机组成。变形器系统具有在奔腾PC上运行的基于Windows的软件。拟议的奥林巴斯显微镜的配置是为了方便其在多用户设施中的操作和四个主要用户的项目。为了满足用户实验室对高分辨率、高灵敏度图像采集以及视频率彩色成像的需求，提出了两个CCD摄像机。应用范围从用DIC或免疫荧光对静态固定切片进行成像，到跟踪活的植物细胞内线粒体的运动，到观察果蝇交配过程中荧光蛋白的转移。计算机接口的设计允许快速图像采集和足够的内存来存储一堆连续的图像，以供进一步处理。图像可以叠加以增强信号或定位荧光抗体在细胞内的位置。显微镜将配备一个Z马达，这样就可以获得连续焦点平面的图像。计算机还可以通过选择适当的滤光片来控制照明波长。显微镜上的快门将由软件控制，以便图像采集可以与它们的打开同步。在可能被光漂白或以其他方式损坏的样品中，这一特征对于在显影过程中通过时间推移观察相同的样品非常重要。Metamorph软件包允许分析对象的许多特征。用户的应用包括测量细胞、细胞核和细胞器的数量，比较不同对象和区域的荧光强度，比较对象在不同细胞或细胞区域中的形状和分布等。一些用户还希望制作植物、果蝇、酵母和线虫的活细胞的录像带电影。该软件包还将用于处理来自现有共焦显微镜的图像，特别是增强其三维重建和形态测量分析的能力。拥有不同项目的四个主要用户计划使用这些设备。用绿色荧光蛋白(GFP)和其他探针标记的植物细胞器的形状、数量、大小、相互作用、融合、分裂、体积和膜电位将在不同的发育时期在不同的组织中进行检测，特别是雄性生殖器官。花粉发育有缺陷的突变体将进行光学切片，以确定发育停滞的阶段。在交配过程中，雌性果蝇将观察到GFP标记的附腺蛋白在雌性果蝇体内的去向。GFP与果蝇核膜蛋白YA的融合将被用来成像活胚胎快速细胞周期中细胞核的存在和移动。线虫缺陷突变体的分离将被用来理解受精后受精卵的极性是如何建立的，以及早期卵裂球中纺锤体取向的差异是如何控制的。这些突变体中的细胞质流动速度将通过跟踪细胞质颗粒的轨迹进行分析。我们将在体内研究与细胞分裂过程中染色体分离有关的果蝇蛋白的定位和分布。染色体准确分离到子代细胞中出现异常的突变体将被描述。

We propose to obtain an Olympus BX-50 microscope equipped with epifluorescence, phase, and DIC optics along with an imaging system capable of acquiring and analyzing high-resolution black-and-white images and lower resolution video rate color images. Specifically, we propose to obtain a MetaMorph Imaging System with a Princeton Instruments Pentamax CCD camera and a Dage MTI three-chip color CCD camera. The MetaMorph system has Windows-based software operated on a Pentium PC. The configuration of the proposed Olympus microscope has been designed to facilitate its operation in a multi-user facility and for the projects of four major users. Two CCD cameras are proposed in order to accommodate the needs of the user labs for both high-resolution, high-sensitivity image acquisition as well as video-rate color imaging. Applications range from imaging of static fixed sections with DIC or immunofluorescence, to following mitochondrial movement within living plant cells, to observing transfer of fluorescent proteins during Drosophila mating. The computer interface is designed to allow rapid image acquisition and sufficient memory to store a stack of sequential images for further manipulation. Images can be superimposed to enhance signals or to locate the position of fluorescent antibodies within cells. The microscope will be equipped with a Z motor so that images of sequential planes of focus can be acquired. The computer can also control the illumination wavelength by selecting the appropriate filter cubes. Shutters on the microscope will be controlled by the software so that image acquisition can be synchronized with their opening. In samples which may be photobleached or otherwise damaged, this feature is important to observe the same specimen during development by time-lapse. The Metamorph software package allows analysis of numerous features of objects. Among the applications of the users are measurement of cell, nuclei, and organelle number, comparison of fluorescen t intensities of different objects and areas, comparison of object shape and distribution in different cells or regions of cells, etc. Some users also wish to produce videotape movies of living cells of plants, Drosophila, yeast, and nematodes. The software package will also be used to process images derived from an existing confocal microscope, particularly enhancing its capability in 3-D reconstruction and morphometric analysis. Four major users with diverse projects plan to use the equipment. Shapes, numbers, size, interactions, fusion, splitting, volume, and membrane potential of plant organelles labelled with green fluorescent protein(GFP) and other probes will be examined in different tissues at various times in development, with particular attention to male reproductive organs. Mutants with defective pollen development will optically sectioned to determine the stage of developmental arrest. The destination of GFP-labelled accessory gland proteins will be observed in the female fruit fly following transfer from the male during mating. GFP fusions with a Drosophila nuclear envelope protein, YA, will be used to image the presence and movement in nuclei during the rapid cell cycles of living embryos. Partitioning defective mutants of C. elegans will be used to understand how the polarity of the zygote is established after fertilization and how the differences in spindle orientations in the early blastomeres are controlled. Cytoplasmic flow rates in these mutants will be analyzed by following the trajectories of cytoplasmic particles. The localization and distribution of Drosophila proteins involved in chromosome segregation during cell division will be examined in vivo. Mutants with aberration in the accurate segregation of chromosomes to daughter cells will be characterized.