Three-Dimensional Fine Structure of Cells and Tissues

Three-Dimensional Fine Structure of Cells and Tissues

Research Emphasis

Current Research

High-voltage electron microscopy (HVEM) offers scientists the possibility of viewing structural details in specimens whose thickness defies imaging by other forms of EM. This permits better three-dimensional (3-D) reconstruction of cellular architecture at resolutions ~40X than can be achieved by light microscopy. Relevant samples include thick sections (200–500 nm) of cells and tissues or isolated organelles, such as chromosomes, chloroplasts, mitotic spindles, and axonemes.

Stereo images can supply some 3-D information, but more detailed data are available from tomograms calculated from multiple tilted views, e.g., 150 images taken over 140º of tilt about each of two orthogonal axes. The resulting reconstructions show resolution of ~6 nm. This facility is also developing technology for the preparation of reliable cellular specimens. High-quality preservation can be achieved through rapid freezing with or without subsequent freeze-substitution fixation. The resource also is working on specific labeling of macromolecular components of cells, both by conventional immunolabeling and by novel means. Methods for tomographic imaging of frozen hydrated samples are also under development.

Resource Capabilities

Instrument

Microscopes available include a 1-MeV HVEM (JEOL and JEM 1000) as well as 300- and 200-KeV instruments (FEI, F30, and F20) with field emission guns that enhance defocus phase contrast in frozen-hydrated specimens. Equipment for specimen preparation includes plunge and high-pressure freezers as well as instruments for freeze substitution, low-temperature embedding, and microtomy at temperatures ranging from ambient to liquid nitrogen. Image-processing computers include six fast personal computers operating under Linux and two older Silicon Graphics Octanes.

Special Features

The usable voltage range for the HVEM is 500B750 KeV. This scope uses side entry operation, a grid diameter of 3 mm, a magnification range of 150B250,000 X, resolution better than 3 Å lattice, routine goniometer stage tilting ±60ºabout any axis, dark field by tilted beam, electron diffraction camera lengths of 1B4 m; and a 1-K lens-coupled CCD camera. The intermediate-voltage microscopes cover 80B300 KeV; both have high-precision goniometer stages and 2-K CCD cameras. A Gatan Imaging Filter will soon be added to the F30, together with a lens-coupled 4-K CCD camera. The lab's image-processing software for 3-D reconstruction on Unix or Linux machines is available for free and may be downloaded from its Web site.