Biomedical Technology Resources
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Visualization of Biological Complexity Resource

Joachim Frank, Ph.D.
P41RR001219
N/A
Retired

Center Overview

The resource develops methods for the three-dimensional (3-D) visualization of biological systems over a wide range of scales, from macromolecular assemblies to cells, using electron microscopy and advanced computational methods. Driven by research projects studying the mitochondrion, kinetochore, calcium release channel, and ribosome, the following three areas of technology and research development are pursued.

Cryoelectron tomography: This center is working to improve tomographic resolution for identification of macromolecular complexes in cells and to extend cryotomographic applications to cells within tissue. Improved imaging during tilt-series collection is achieved by means of electron microscope automation, energy filtering, and advanced phase-contrast techniques. Computational methods are being developed to enhance reconstruction fidelity and to aid in visualization and extraction of macromolecular and organellar motifs. Study of 3-D subcellular structure in frozen-hydrated tissue is facilitated by developments in cryoultramicrotomy and cryofocused ion-beam milling, which are carried out in collaboration with Rensselaer Polytechnic Institute (see below) and Dr. Eric Lifshin, Metrology and Characterization Center, Albany NanoTech, University at Albany.

Time-resolved cryoelectron microscopy: Time-resolved imaging of macromolecular interactions, using single-particle reconstruction methodology, enables the understanding of physiologic function from 3-D snapshots of a dynamic system. The goal is to develop methods to initiate reactions involving macromolecules, trap them in intermediate states, and visualize them using cryoelectron microscopy. This work is aided by collaboration with scientists at Rensselaer Polytechnic Institute: Dr. Omkaram Nalamasu and J. Jay McMahon (Center for Integrated Electronics), Dr. Toh-Ming Lu (Department of Applied Physics), and Dr. Pulickel Ajayan (Department of Materials Science and Engineering). The center also collaborates with Dr. Howard White of the Eastern Virginia Medical School in the development of a computer-controlled time-resolved plunge-freezing machine.

Classification of heterogeneous data in single-particle reconstruction: The reconstruction of macromolecular complexes from thousands of low-dose images that show the molecule in different views makes the tacit assumption that the molecule exists in the specimen in the same conformation or binding state. In practice, this assumption is often not fulfilled. In those cases, the reconstruction is blurred and fails to reflect the true structure of any of the conformers. This center is systematically exploring methods capable of sorting heterogeneous molecule populations, so that subpopulations can be found and separately reconstructed. This work is being done in collaboration with Dr. Jose-Maria Carazo of Universidad Autonoma de Madrid, Dr. Gabor Herman of the City University of New York, Dr. Harry Zuzan of McGill University, and Dr. Pawel Penczek of the University of Texas, Houston.