ENHANCEMENT OF MICROSCOPE & ITS COMPUTER CONTROL

显微镜的增强

• 批准号: 6354268
• 负责人: GARY FAN
• 金额: $ 7.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6354268
• 关键词: bioengineering /biomedical engineering; biomedical equipment development; biomedical resource; biotechnology; building /facility design /renovation

Aim 1: Enhancement of the Microscope and its Computer Control The objective of this core TR&D project is to develop and improve EM techniques for image contrast enhancement and 3D information extraction from biological specimens, particularly thick sections, with computer-controlled specimen positioning, electron optics and a use of a direct digital readout image acquisition system being developed under a separate subproject (see following project description). Progress has been made in the following areas: Computer control and microscope automation: A microscope control library has been established and new routines are added as need arises. All optical parameters and most mechanical controls (such as the four axes of the stage) can now be remotely controlled by a local host as well as by workstations linked to the host by the computer network. Semi-automated tomography data acquisition software has been developed and used successfully for film-based and slow-scan CCD camera-based tomography. In addition, we have implemented an automated specimen survey capability utilizing stage movement. A large mosaic of 10 x 10 or more 1k x 1k images can be collected automatically, covering a few square mm. This montage is sent to the remote user before a telemicroscopy session and serves as a low magnification map for selecting potentially interesting regions to explore at higher magnifications. Characterization of the high tilt coil-enabled optical sectioning possibilities with IVEM: During the previous year JEOL finally completed the installation of a new set of image shift coils above the mini-lens assembly, below the objective lens. Delivery of these long-awaited coils allowed us to begin to test the final part of our scheme to develop electron optical sectioning by creating a digital rotary hollow-cone illumination above the specimen.. Briefly we first developed a unique electron optical design for this IVEM which enabled operation in the so-called "B" mode. This new optical scheme has several advantages, some of which were noted above:  It provides for high contrast, high resolution imaging of thick sections due to the removal of multiply scattered electrons and an energy filtering effect.  It enables on-line 3D observation and stereo views may be displayed quickly with tilts on any azimuth as the method uses beam tilt instead of stage tilt;  It provided us with a test-bed for experimentation toward the development of a fully functional "optical sectioning" electron microscope. This work involved the use of specially designed scan and descan coils to form a synchronized hollow-cone illumination to reduce depth of field. The new coils seem to be perfect and preliminary data demonstrating optical sectioning capabilities has now been obtained. Addition of confocal and 2-photon microscopes: Although not specifically described as a technology aim in the original NCMIR proposal to NCRR , the availability of advanced light microscopes are essential to the activities of the resource for performing correlative light and electron microscope 3D analyses. Recognizing this we arranged for BioRad and Nikon to become research partners with NCMIR and to donate upgrades or provide instruments for our use. During the current period of funding, we upgraded the older Biorad 600 laser-scanning confocal housed in the NCMIR (originally purchased in 1989 for specific research projects by Ellisman and Terry) to a new 24bit 1024 system. This was done through a generous partnering arrangement with BioRad at no cost to the Resource. In addition, Nikon Corporation (Japan) established a research agreement (funded by a grant to Ellisman and UCSD) which included a donation of a Nikon RCM8000 video-rate confocal system . This system has now been modified by us to provide both UV or 3 channel visible light high speed confocal imaging as well a high speed multi-photon imaging. The microscope, incorporates a femto-second, tunable, pulsed laser providing excitation at wavelengths from 690 to 1050 nm, an NCMIR designed and constructed pre-chirper optical system for laser pulse-width compression, and a non-confocal detection assembly. A 75% increase in fluorescent emission is consistently obtained with the use of the prechirper optics. The non-confocal assembly is capable of detecting the ratio of two emission wavelengths and provides a 125% increase in detection efficiency compared to confocal detection. Ratio imaging and optical sectioning can therefore be performed more efficiently using multiphoton imaging than with confocal optics. Useful two-photon images can be acquired at video rate with a laser power as low as 2.7 mW at the specimen using genetically modified green fluorescent proteins (GFPs). To our knowledge, this is the first system to deliver video rate (and faster) 2-photon images of living preparations. We recently succeeded in acquiring images of moving GFP-labeled bacteria at rates of 1 frame/4msec. (32x512 pixels). This instrument will be further modified for use in at least one of the correlated microscopy projects with David Kleinfeld's group proposed in our pending NCRR renewal application.

目标 1：增强显微镜及其计算机控制 该核心 TR&D 项目的目标是开发和改进 EM 图像对比度增强和 3D 信息技术 从生物样本中提取，特别是厚切片， 具有计算机控制的样品定位、电子光学和 使用直接数字读出图像采集系统 在一个单独的子项目下开发（参见以下项目 描述）。 在以下方面取得了进展： 计算机控制和显微镜自动化：显微镜控制 库已建立，并根据需要添加新例程 出现。 所有光学参数和大多数机械控制（例如 舞台的四个轴）现在可以由本地远程控制 主机以及通过计算机链接到主机的工作站 网络。 半自动断层扫描数据采集软件 开发并成功用于基于胶片的慢扫描 CCD 基于相机的断层扫描。 此外，我们还实施了 利用载物台移动的自动化样本测量能力。 一个 可以收集 10 x 10 或更多 1k x 1k 图像的大马赛克 自动覆盖几平方毫米。 该剪辑被发送至 远程显微镜会话之前的远程用户，并充当低 用于选择可能感兴趣的区域的放大图 以更高的放大倍率进行探索。 高倾斜特性 使用 IVEM 实现线圈光学切片的可能性： 去年JEOL终于完成了一套新的安装 图像移动线圈位于微型镜头组件上方、物镜下方 镜片。 这些期待已久的线圈的交付使我们能够开始 测试我们开发电子光学计划的最后部分 通过在上面创建数字旋转空心锥照明来进行切片 标本.. 简而言之，我们首先开发了一种独特的电子光学 该 IVEM 的设计可在所谓的“B”中运行 模式。 这种新的光学方案有几个优点，其中一些 如上所述：它提供高对比度、高分辨率 由于去除了多重散射而对厚切片进行成像 电子和能量过滤效应。 它支持在线 3D 观察和立体视图可以通过倾斜快速显示 任何方位角，因为该方法使用波束倾斜而不是平台倾斜；它 为我们提供了一个试验台来进行开发实验 功能齐全的“光学切片”电子显微镜。 这 工作涉及使用专门设计的扫描和去扫描线圈 形成同步空心锥照明以减少景深。 新线圈似乎很完美，初步数据表明 现在已经获得了光学切片能力。 添加 共焦和 2 光子显微镜：虽然没有具体说明 在向 NCRR 提交的原始 NCMIR 提案中被描述为技术目标， 先进的光学显微镜的可用性对于 用于执行相关光和资源的活动 电子显微镜 3D 分析。 认识到这一点，我们安排了 BioRad 和尼康将成为 NCMIR 的研究合作伙伴并捐赠 升级或提供仪器供我们使用。 当前期间 在资助期间，我们升级了旧的 Biorad 600 激光扫描仪 位于 NCMIR 中的共焦（最初于 1989 年购买，用于 Ellisman 和 Terry 的具体研究项目）到新的 24 位 1024 系统。 这是通过慷慨的合作安排完成的 BioRad 不收取资源费用。 此外，尼康公司 （日本）建立了一项研究协议（由赠款资助） Ellisman 和 UCSD），其中包括捐赠一台尼康 RCM8000 视频速率共焦系统。 该系统现已被我们修改 提供 UV 或 3 通道可见光高速共焦 成像以及高速多光子成像。 显微镜， 结合了飞秒、可调谐、脉冲激光器，提供 激发波长为 690 至 1050 nm，NCMIR 设计并 构建激光脉冲宽度预啁啾光学系统 压缩和非共焦检测组件。 增加了 75% 荧光发射始终如一地获得与使用 预啁啾光学器件。 非共焦组件能够检测 两个发射波长的比率，并提供 125% 的增加 与共焦检测相比的检测效率。 比率成像 因此可以更有效地进行光学切片 使用多光子成像优于共焦光学。 有用 双光子图像可以以视频速率获取，激光功率为 使用转基因绿色样品的功耗低至 2.7 mW 荧光蛋白（GFP）。 据我们所知，这是第一个 系统提供视频速率（和更快）的活体 2 光子图像 准备工作。 我们最近成功获取了移动的图像 GFP 标记的细菌，速率为 1 帧/4 毫秒。 （32x512 像素）。 该仪器将被进一步修改以用于至少一种 与 David Kleinfeld 小组的相关显微镜项目 在我们待决的 NCRR 续签申请中提出。