CORE--CONFOCAL MICCROSCOPY FACILITY

传统光学显微镜的理想标本是二维的。 然而，生物材料是在三个维度上组织的。vbl.使用 传统的荧光显微镜，所有的荧光都产生在 生物标本的深度达到图像平面。因此， 焦平面内的材料被清晰成像，而焦平面外的材料被清晰成像 焦平面的变化会产生对比度降低的背景。这个问题是 用共聚焦显微镜绕过。用共聚焦显微镜，光 来自激光照射的针孔聚焦在某个点上 在一个物体里。来自同一点的荧光随后被成像 使用探测器针孔。因为照明针孔和背面- 检测针孔的投影在对象中具有共同焦点， 只有从样品产生的光才能到达探测器 层，并且几乎所有的散焦荧光都从 图像。使用共聚焦显微镜，我们可以解决摄取反义DNA 通过癌细胞，细胞器在细胞分裂过程中的运动和遗传， 肿瘤诱导剂对蛋白质亚细胞定位的影响 以及细胞建立过程中细胞骨架组织的变化 极化、细胞迁移和细胞分裂。 1997年1月，Liza Pon博士成为癌症中心的新主任 共焦显微镜设备。她介绍了一种最先进的共焦 使用从共享仪器赠款获得的资金的成像系统。 新的设施现在被癌症中心大量使用(大约每天6小时) 成员和其他研究科学家。该系统由蔡司LSM组成 安装在蔡司Axiovert 100电视上的410扫描激光共焦附件 倒置荧光显微镜。样品激发和共焦成像 探测是使用一台氩-氪激光器和三个高度 灵敏的光电倍增管探测器。该系统最高可成像到 同时三个荧光团，并获得差分干涉 对比度(DIC)和相位对比度图像。该软件包适用于 共焦图像分析提供三维重建， 三维图像的立体显示、延时成像、 比率成像、光漂白(例如，FRAP)、量化共定位 和形态测量学。共焦显微镜与倒置显微镜的结合 显微镜和数字图像分析让用户可以观察复杂 活的或固定的系统，比以往任何时候都更准确和更快。

The ideal specimen for conventional optical microscopy is two-dimensional. However, biological material is organized in three dimension. Using conventional fluorescence microscopy, all fluorescence generated over the depth of a biological specimen reaches the image plane. As a result, the material within the plane of focus is sharply imaged, and material outside of the focal plane produces contrast-reducing background. This problem is circumvented using confocal microscopy. With confocal microscopy, light originating from a laser-illuminated pinhole is focused on a certain point in an object. Fluorescence from the same point is subsequently imaged using a detector pinhole. Since the illumination pinhole and the back- projection of the detection pinhole have a common focus in the object, the only light that reaches the detector is that generated from the specimen layer, and virtually all out-of-focus fluorescence is eliminated from the image. Using confocal microscopy, we can resolve uptake of antisense DNA by cancer cells, organelle motility and inheritance during cell division, the effect of the tumor inducers on subcellular localization of proteins, and changes in cytoskeletal organization during establishment of cell polarization, cell migration and cell division. In January 1997, Dr. Liza Pon became the new director of the Cancer Center Confocal Microscopy Facility. She introduced a state-of-the-art Confocal Imaging System using funds obtained from a Shared Instrumentation Grant. The new Facility is now heavily used (ca. 6 hours/day) by Cancer Center members and other research scientists. The system consists of a Zeiss LSM 410 scanning laser confocal attachment mounted on a Zeiss Axiovert 100 TV inverted fluorescence microscope. Sample excitation and confocal image detection is accomplished using an argon-krypton laser and three highly sensitive photomultiplier detectors. This system is able to image up to three fluorophores simultaneously, and to obtain differential interference contrast (DIC) and phase contrast images. The software package for confocal image analysis offers three-dimensional reconstruction, stereoscopic display of three-dimensional images, time-lapse imaging, ratio imaging, photobleaching (e.g., FRAP), quantifying co-localization and morphometry. The combination of confocal microscopy with the inverted microscope and digital image analysis allows users to observe complex living or fixed system with greater accuracy and speed than ever before.