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.
