ENHANCEMENT OF MICROSCOPE & ITS COMPUTER CONTROL

显微镜的增强

• 批准号: 6469017
• 负责人: GARY FAN
• 金额: $ 10.66万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6469017
• 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项目的目的是开发和改善EM 图像对比度增强和3D信息的技术 从生物标本，尤其是厚部分中提取 具有计算机控制的标本定位，电子光学和A 使用直接数字读数图像采集系统的使用 在单独的子标题下开发（请参阅以下项目 描述）。 在以下领域取得了进展： 计算机控制和显微镜自动化：显微镜控制 已经建立了库，并根据需要添加新的例程 出现。 所有光学参数和大多数机械控件（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。 半自动化层析成像数据采集软件已经 成功地用于基于电影和慢速的CCD 基于摄像机的断层扫描。 此外，我们已经实施了 使用舞台运动的自动标本调查能力。 一个 可以收集10 x 10或更多1K x 1k图像的大镶嵌物 自动覆盖几毫米。 这个蒙太奇被发送到 远程用户在远程镜检查之前，并将其作为低 用于选择潜在有趣区域的放大图 在更高的宏伟探索中探索。 高倾斜的特征 IVEM的启用线圈的光学切片可能性：在此期间 杰尔（Jeol）终于完成了一套新的安装 在物镜下方的迷你镜头组件上方的图像移动线圈 镜片。 这些期待已久的线圈的交付使我们开始 测试我们开发电子光学方案的最后一部分 通过在上面创建数字旋转空心孔照明来分割 标本。简短地，我们首先开发了独特的电子光学 该IVEM的设计，该IVEM在所谓的“ B”中启用了操作 模式。 这个新的光学方案具有几个优势，其中一些 上面指出：它提供了高对比度，高分辨率 由于去除倍数散射而导致的厚部分的成像 电子和能量过滤效果。 它可以在线3D 观察和立体声视图可以很快显示出倾斜度 任何方位角作为该方法使用横梁倾斜而不是舞台倾斜；它 为我们提供了用于开发实验的测试床 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DESCAN线圈 形成同步的中空光照明，以减少景深。 新的线圈似乎是完美的，初步的数据证明了 现在已经获得了光学切片功能。 加法 共聚焦和2光子显微镜：尽管不是具体 被描述为针对NCRR的原始NCMIR提案的技术， 高级光显微镜的可用性对于 用于执行相关光的资源活动和 电子显微镜3D分析。 认识到我们安排的 Biorad和Nikon成为NCMIR的研究合作伙伴并捐赠 升级或提供我们使用的工具。 在电流期间 资金时期，我们升级了较旧的Biorad 600激光扫描 共聚焦在NCMIR（最初于1989年购买） Ellisman和Terry的特定研究项目）到新的24位1024 系统。 这是通过与 无需花费资源。 此外，尼康公司 （日本）建立了一项研究协议（由 Ellisman和UCSD），其中包括捐赠Nikon RCM8000 视频速率共聚焦系统。 该系统现在已被我们修改 提供紫外线或3通道可见光高速共焦 成像以及高速多光子成像。 显微镜， 合并一个femto秒，可调，脉冲激光器提供 在690至1050 nm的波长下激发，设计的NCMIR和 用于激光脉冲宽度的手术前光学系统 压缩和非相互检测组件。 增长75％ 通过使用 prechirper光学元件。 非相关组件能够检测 两个排放波长的比率，可增加125％ 与共聚焦检测相比，检测效率。 比率成像 因此，可以更有效地执行光学切片 使用多光子成像比共聚焦光学元件。 有用 可以以视频速率以激光功率作为视频速率获取两光子图像 在样品处使用转基因绿色的标本低至2.7兆瓦 荧光蛋白（GFP）。 据我们所知，这是第一个 提供视频速率（和更快）的2张照片的系统 准备。 我们最近成功地获取了移动的图像 GFP标记的细菌以1帧/4msec的速率。 （32x512像素）。 该仪器将被进一步修改，以在至少一种 与David Kleinfeld小组相关的显微镜项目 在我们未决的NCRR续订申请中提出。