High Resolution Microdensitometer for Structural Biology

用于结构生物学的高分辨率显微密度计

• 批准号: 9512940
• 负责人: David DeRosier
• 金额: $ 11.93万
• 依托单位: Brandeis University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9512940&HistoricalAwards=false
• 关键词: Resolution; Microdensitometer; Structural; Biology

In this application we describe research programs in structural biology at two different institutions, each of which requires the ability to analyze data obtained from our high resolution electron microscopes. In order to be able to fully utilize the capabilities of our EM Facilities and to overcome a major gap in the technological resources available to the life sciences communities at Brandeis University and at Boston University School of Medicine, we are proposing to purchase a high resolution precision microdensitometer scanning system. Considerable effort has been devoted to insure that the Rosenstiel Basic Medical Sciences Research Center (the Center) and BUSM, Department of Biophysics have the EM facilities necessary to pursue high-resolution structural studies. Over the past few years several projects have developed specimens extremely well suited for Cystallographic and single particle study using EM techniques. Optical diffraction of film recorded EM images and/or direct observation of film recorded electron diffraction patterns of a variety of these specimens have indicated that the information content of much of this data is great and that Programs for high-resolution structural determination are feasible. Central to working with any film recorded data is the ability to convert this information into digital form for computer-based processing and analysis. In neither institution is there currently a device able to digitize EM film recorded data in a form suitable for high-resolution structural studies. (The digitization equipment currently available is inadequate even for moderate resolution studies.) The few EM based high-resolution reconstructions performed on biological samples (bacteriorhodopsin, light harvesting complex and porin) in other laboratories were exclusively determined with data (images and diffraction patterns) digitized through precision microdensitometers. Both the Center's high resolution electron microscopes (Philips CM12 and Model 420) are routinely providing high resolution images for our structural biology research programs. However, in order to digitize these images for computer analysis it has been necessary for our investigators to travel to Albany, NY as there are no suitable scanners in New England. The instrument of choice for this application is currently being manufactured by Orbital Sciences Corporation (Pomona, CA). It is a flat-bed microdensitometer and is essentially identical to the Perkin-Elmer instrument which is no longer manufactured. It is widely regarded as the state-of-the-art instrument for image digitization and far exceeds the capabilities of our current scanner. The technique used in the Orbital microdensitometer system is almost completely free of optical aberrations and produces high fidelity data. The major advantage of this instrument is that an illuminated area of the specimen is optically projected onto a sample defining aperture as small as 3~m. Only that light which passes through the aperture is measured and digitized. Scanning is accomplished by laterally translating the specimen relative to the optical axis. The aperture maintains a fixed location on the optical axis, thereby minimizing distortions and the effects of optical aberrations. The Center's only available instrument is an Eikonix digitizer which operates by projecting a real image of the specimen to be digitized using a 35mm photographic lens. This real image is digitized via a linear photodiode array which is translated across the image plane. This approach, though relatively inexpensive, is subject to a multitude of optical and mechanical defects. The Eikonix scanner cannot support our high resolution structural studies reliably.

在本申请中，我们描述了两个不同机构的结构生物学研究项目，每个项目都需要能够分析从我们的高分辨率电子显微镜获得的数据。为了能够充分利用我们的EM设施的能力，并克服布兰迪斯大学和波士顿大学医学院生命科学界可用的技术资源的重大差距，我们建议购买一个高分辨率精密显微密度扫描系统。 已作出相当大的努力，以确保 罗森斯蒂尔基础医学科学研究中心（Rosenstiel Basic Medical Sciences Research Center） 中心）和BUSM，生物物理系有EM 追求高分辨率结构所需的设施 问题研究在过去的几年里， 开发出的标本非常适合 使用EM的晶体学和单颗粒研究 技术.胶片记录电磁图像的光学衍射 和/或直接观察胶片记录的电子 各种各样的这些标本的衍射图案 表明其中大部分数据的信息内容 是伟大的，高分辨率结构的程序， 决心是可行的。 处理任何胶片记录数据的核心是将这些信息转换为数字形式的能力 用于计算机处理和分析。既不在 机构目前是否有一种设备能够 以适合于高分辨率的形式记录的胶片数据 结构研究。(The数字化设备目前 即使对于中等分辨率也是不够的 研究。）少数基于EM的高分辨率重建 对生物样品（细菌视紫红质、光 在其他实验室中收获复合物和孔蛋白）是独家的 通过数据（图像和衍射）确定 图案）通过精确数字化 显微密度计该中心的高分辨率电子显微镜（飞利浦CM 12和 模型420）是例行提供高分辨率的图像，为我们的结构生物学研究 程序.然而，为了将这些图像进行计算机分析， 我们的调查人员有必要前往纽约州的奥尔巴尼，因为纽约州没有合适的扫描仪。 英格兰该应用的首选工具目前正在由 轨道科学公司（加利福尼亚州波莫纳）。它是一种平板显微密度计， 与不再生产的Perkin-Elmer仪器相同。它被广泛认为 作为最先进的图像数字化仪器， 当前扫描仪该技术用于 轨道显微密度计系统几乎 完全没有光学像差，并产生高保真数据。的主要优点 该仪器的一个优点是将样品的照明区域光学投影到样品上 孔径小至3 μ m。只有通过孔径的光才被测量 数字化。扫描是通过相对于样品横向平移样品来完成的。 光轴光圈在光轴上保持固定位置，从而最小化 扭曲和光学效应 失常。 该中心唯一可用的仪器是 Eikonix数字化仪，其通过投影待数字化的样本的真实的图像来操作， 35毫米摄影透镜。该真实的图像通过线性光电二极管阵列数字化， 在图像平面上平移。这 这种方法虽然相对便宜，但受到 许多光学和机械缺陷。 Eikonix扫描仪无法支持我们的高 可靠的分辨率结构研究。