High Resolution Microdensitometer for Structural Biology

在本申请中，我们描述了两个不同机构的结构生物学研究计划，每个机构都需要分析从我们的高分辨率电子显微镜获得的数据的能力。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主要缺口，我们提议购买高分辨率精密显微密度计扫描系统。为确保罗森斯蒂尔基础医学研究中心(该中心)和BUSM生物物理系拥有进行高分辨率结构研究所需的EM设施，已经付出了相当大的努力。在过去的几年里，几个项目已经开发出非常适合于晶体相学和使用EM技术进行单粒子研究的标本。薄膜记录的EM图像的光学衍射和/或对各种样品的薄膜记录的电子衍射图的直接观察表明，这些数据中的许多数据的信息量很大，高分辨率结构确定程序是可行的。处理任何胶片记录的数据的核心是将这些信息转换为数字形式以便基于计算机的处理和分析的能力。在这两个机构中，目前都没有能够将EM胶片记录的数据以适合高分辨率结构研究的形式数字化的设备。(即使对于中等分辨率的研究，现有的数字化设备也是不够的。)在其他实验室的生物样品(细菌视紫红质、捕光复合体和孔蛋白)上进行的少数基于EM的高分辨率重建是通过精密显微密度计数字化的数据(图像和衍射图)专门确定的。该中心的高分辨率电子显微镜(飞利浦CM12和420型)通常都为我们的结构生物学研究项目提供高分辨率图像。然而，为了将这些图像数字化以进行计算机分析，我们的调查人员必须前往纽约州的奥尔巴尼，因为新英格兰没有合适的扫描仪。用于这一应用的选择仪器目前正在由轨道科学公司(加利福尼亚州波莫纳)制造。它是一种平板显微密度计，与已不再生产的Perkin-Elmer仪器基本相同。它被广泛认为是最先进的图像数字化仪器，远远超过我们目前扫描仪的能力。轨道显微密度计系统中使用的技术几乎完全没有光学像差，并产生高保真数据。这种仪器的主要优点是，样品的照明区域被光学投影到定义了小到3米的孔径的样品上。只有穿过孔径的光被测量和数字化。扫描是通过相对于光轴横向平移样品来完成的。该孔在光轴上保持固定位置，从而使失真和光学像差的影响最小化。该中心唯一可用的仪器是Eikonix数字化仪，它使用35 mm的摄影镜头投射要数字化的标本的真实图像。该真实图像通过在图像平面上平移的线性光电二极管阵列来数字化。这种方法虽然相对便宜，但存在大量的光学和机械缺陷。Eikonix扫描仪不能可靠地支持我们的高分辨率结构研究。

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.