Hans Ris - US grants

The High Voltage Electron Microscope Facility is a Biotechnology Resource of the Biotechnology Resources Branch of N.I.H. and is available to bio-medical researchers for ultrastructural studies where use of a high voltage electron microscope can provide new information. Investigations are directed especially towards analysis of relatively large and complex three-dimensional cell structures either in intact cells or isolated organelles, such as isolated flagella, chromosomes and Golgi complexes, or in sections up to 1 or 2 microns thick. The cold stage will undergo further development and modification. Freezing and cold sample handling procedures have been developed for viewing frozen sections, whole specimens, isolated cell components and aqueous suspensions. An improved type of image intensifier and digital image/memory processor has been installed and successfully used. This is part of a program to improve resolution and performance at high magnification in order to make possible high resolution studies of macromolecular crystals. The H.V.E.M. is particularly well suited to high resolution studies of this type because of its greater penetrating power, its greater depth of field and because of its greater specimen chamber size, one sufficiently large to permit introduction of ancillary equipment such a tilting/cooling stages which are necessary to preserve and orient these biological structures. Methods are being developed for immunocytochemical labeling of cell structures such as cytoskeletal elements, both in cell whole mounts and in thick sections.

An apparatus has been designed that allows light microscopic observation of living cell phenomena immediately followed by fast immobilization of these phenomena with the aid of rapid freezing. Then the ultrastructure of this cell cryo-whole-mount can be observed in an electron microscope using a cryo-stage. This apparatus consists of phase contrast, epifluorescence, and polarized light optical systems assembled onto an optical bench. These optical systems are integrated into one unit with a specimen carrier assembled onto a sliding mechanism delivering the specimen into melting ethane. The unique feature of this apparatus is that it allows us to accomplish the fundamental goal of ultrastructural studies in life sciences, i.e. direct correlation between images of a living cell with images of this cell's ultrastructure, thus direct interpretation of molecular mechanisms in a living cell. Using an apparatus prototype, we were able to capture early events involved in adhesion of metastasizing cancer cells. Furthermore, we have been able to discover a very delicate intranuclear cable network that probably serves as a part of the cell distribution or signalling system. The goal of this project is to build a permanent instrument, to refine its operation, and to prepare its expansion toward new imaging capabilities by making six improvements: (1) addition of an intensified charge-coupled device (ICCD) camera to allow observations on living cells labelled with specific probes at low luminance; (2) addition of a filter-wheel containing fluorochrome excitation and infrared filters, as well as a multi-band barrier filter to ensure the correct register of images during recording of images of cells labelled with multiple probes; (3) addition of a dedicated computer to control shutters and sliders and to link the apparatus to EtherNet communication; (4) addition of Nomarski optics and motorized specimen stage to provide us with the capability of optical s ectioning; (5) preparation for the attachment of a real-time confocal laser-scanning imaging system and optical tweezers; (6)preparation for the attachment of an atomic force microscope (AFM). Development of the proposed instrument is now essential to ensure progress in four research projects; (a) structure and function of the nuclear pore complex; (b) myofibril assembly in mammalian skeletal muscle and the role played by expression of titin gene for this assembly, (c) actomyosin based motility of keratocytes; (d) reduction in Rhabdomyosarcoma malignancy by forced expression of myoD, myogenin and titin genes.

microscopy; human tissue; growth factor; biomedical resource;