Digital three‐dimensional visualization of cellular organelles studied by medium‐ and high‐voltage electron microscopy

Abstract

K E Y w ORDS. Medium-and high-voltage EM, nucleolus, Ag-NOR, bas-relief. S U M M A R YTilted thick sections (one-half to several micrometres) of biological specimens observed with medium-to high-voltage electron microscopes are extremely useful for the study of the three-dimensional (3-D) structure of organelles.If high resolution in 3-D visualization and 3-D reconstruction is needed, many images corresponding to various angles of rotation and tilt must be recorded. This necessitates very time-consuming work-including eventual photographic processing-before good positioning of the object is defined.We have developed software which permits very rapid and precise determination of the tilt-axis, the registration of tilted views, 3-D measurements and 3-D visualization. Images are digitized either from negative films or directly with a camera fitted to the microscope. The application of the software is performed in minutes and allows for a rapid check of the quality of the tilt-series and of the features of interest of the object.Application of the software to the study of the 3-D structure of active components of the nucleolus stained with silver is shown.

Three‐dimensional imaging of the mucus secretory process in the cryofixed frog respiratory epithelium

“…In the literature, it is generally admitted that the goblet cells shape differs from the serous cells in that their secretory granules are electron-lucent, more or less confluent. For the goblet cells of the intestine and sublingual glands of the rat [3] it has been reported that the respiratory mucus is discharged from the mucous cells through gaps formed by the rupture of their apical plasma membrane. The fact is that in electron-dense surface secretory cells (generally called serous cells), the membrane of their secretory granules often appears to be retained without any fusion [19].…”

“…The method of bas-relief micrography has been reported to be particularly suitable to enhance structures with blurred or smeared boundaries like limiting membranes [8]. The improvement of this method [3] gives a standardized 3-D computerized vision of the organelles of the epithelial ciliated and secretory cells by the superimposition of the light and shadows of the added highlights. The main advantage is that this 3-D information obtained from the stereopair can be transferred into a single image easily readable.…”

Chromatin conformation and salt-induced compaction: three-dimensional structural information from cryoelectron microscopy.

“…Stereology (Beorchia et al, 1991;Dustin et al, 1991;Furrer et al, 1995) was used to determine 3-D locations of features in the tilt pairs from which coordinates of nucleosome positions and internucleosomal distances and angles were derived. Where possible, x,y,z coordinates were determined for nucleosome centers and linker DNA entry-exit sites, and several points along each linker DNA and used to calculate the following parameters (see Fig.…”

“…An aqueous environment is retained, and, for structures of the size scale of small oligonucleosomes, the complete 3-D conformation may be preserved. From tilt pairs of micrographs, stereology (Beorchia et al, 1991;Dustin et al, 1991), provides 3-D information concerning distances between consecutive nucleosomes, linker DNA trajectories, and the relative orientation of consecutive nucleosomes. We show here that cryoelectron microscopy provides greatly improved images of oligonucleosomes in solution, allowing the results of solution studies on saltinduced compaction to be correlated directly with changes in conformation.…”