The inner dynein arm complex: compatible images from freeze-etch and thin section methods of microscopy.

A study has been made of the inner dynein arm complex in the sperm flagellum of Gallus domesticus. It has been found that images of the complex made from highly contrasted, approx. 20 nm, sections are very largely compatible with images made from replicas of rapidly cryofixed material. This suggests that neither technique seriously distorts the native state. From both types of image, we conclude that the most proximal group of inner dynein arm heads (IDA 1) is related to spoke S1 and consists of 3 heads with fine connections to the B-tubule. IDA 2 consists of 2 such heads and is related to spoke S2. IDA 3 is apparently single headed and lies close to the A-attachment of spoke S3. This arrangement of IDAs repeats every 96 nm. Between the IDAs and outer dynein arms (ODAs), lying close to IDAs 2 and 3, is a strap-like linkage to the next B-tubule; it is argued that this represents the circumferential link, nexin. In sections, but not consistently in replicas, IDA2 lies closer to the plane of the nexin link than does IDA 1. In the presence of ATP and vanadate, little change is seen in the IDA complex except for an ill-defined alteration to IDA 1. It is speculated that the apparently smaller size of the inner arm heads (compared with the ODA major subunit) may have functional implications in relation to maximal sliding velocity.

Architecture of the outer arm dynein ATPase in an avian sperm flagellum, with further evidence for the B-link.

Demembranated sperm flagella from Gallus domesticus have been prepared by the rapid-freeze, deep-etch, rotary replica technique in order to study the three-dimensional morphology of the outer dynein arm (ODA)-ATPase complex. In general, the ODAs resemble most closely those from the sea urchin Strongylocentrotus described by W. S. Sale et al. In the 'rigor' condition, the ODA consists of a major subunit (the head), from which a slender link extends to the adjacent B-tubule (the B-link). A smaller, intermediate subunit lies adjacent to the head, distally, and a further extension of the complex, the minor subunit, continues distally beneath the head domain of the next arm complex, where it attaches to the A-tubule. In the presence of ATP and vanadate ('relaxed' condition), the attachment point of the B-link to the head is shifted to a more proximal position, and the minor subunit is no longer visible. This is interpreted as resulting from a rotation of the head. These features are demonstrated stereoscopically, and from several viewpoints. Image enhancement has been used to clarify and define the repetitive features of the dynein arrays. In addition, some of the axonemes have been imaged from highly contrasted 20 nm thin sections; the detection of B-links in such sections means that these slender structures cannot be considered artefacts of the rapid-freeze, deep-etch protocol.

Application of x-ray microtomography in materials science illustrated by a study of a continuous fiber metal matrix composite.

The advantages of the use of x-ray microtomography in materials science are discussed, and illustrated by the nondestructive study of the mechanical damage in a continuous fiber SiC/Al composite at a resolution of about 25 µm. A laboratory x-ray source was used, and it was shown that quantitative measurements of the linear absorption coefficient at this resolution are possible, even though the AgKα radiation used is accompanied by a considerable amount of white radiation, provided that the counter system is properly corrected for pulse pile up and dead-time.

Three-dimensional X-ray microscopy with accurate registration of tomographic sections.

X-ray microtomography has been used to study the internal flaws and external shape of a 0.75 X 1 mm copper sulphate crystal at a resolution of 25 microns. The problems of accurate tomographic reconstruction and the subsequent registration of the reconstructed sections to give a complete 3-D image are considered when the position of the axis of rotation of the specimen varies slightly between projections.

X-ray microtomography of biological tissues using laboratory and synchrotron sources.

X-ray microradiography is a well established technique for the study of biological structures in which the projected absorption is measured, usually with photographic film or resist. If scanning X-ray microradiography with a 15-µm beam, 2-D scanning, and photon counting is used, more accurate results can be obtained and real-time experiments undertaken. Addition of a rotation axis allows computerized axial tomography to be done at a resolution of 15 µm. This technique overcomes the inherent difficulty of microradiography that all detail perpendicular to the plane of the specimen is superimposed. This method has been applied to the study of the 3-D mineral distribution in a 0.8×0.8 mm column of human cortical bone with a laboratory X-ray source. Calculation of the wavelength dependence of the linear absorption coefficient for liver and bone shows that, for a choice of wavelength in the range of 3-0.4 Å (4-30 keV), the specimen thickness can be from 100µm-2 cm and 10 µm-3 mm, respectively.Synchrotron X-radiation has the potential for better resolution because of the higher intensity, which allows the use of a narrower beam. There is also the possibility of determining individual element 3-D distributions from measurements on either side of the absorption edges because of the continuous nature of the spectrum and also the possibility of doing this from X-ray fluorescence measurements. To investigate these possibilities, a tomographic apparatus has been built based on the availability of accurately ground, tungsten carbide balls. Metrological assessment shows that the specimen remains within <1 µm of the required position during translation and rotation. Preliminary X-ray tomographic studies with a 4-µm diameter beam have been started at the Daresbury laboratory synchrotron source.

X-ray microscopy using computerized axial tomography.

Computerized axial tomography with MoK alpha X-radiation has been used to study a 0.8 X 0.8 mm column of human femoral bone at a resolution of 15 micron. This non-destructive X-ray microscopic technique allowed 'sections' to be made with 25 micron separation and the distribution of linear absorption coefficient in each section to be determined.

Modeling Balbiani Ring gene transcription with electron microscope tomography.

Cross-sectional views of mature regions of Balbiani Ring (BR) transcription loops were reconstructed by electron microscope tomography. Balsa wood models were built based upon the resulting tomograms. Coordinates of the centers of the ribonucleoprotein BR granules were estimated and employed to calculate the approximate orientation of the BR transcription unit axis in the plastic section. The density of BR granules per micron of transcription unit axis was estimated. Employing structural parameters for mature regions of BR transcription loops derived from present and previous studies, theoretical calculations were performed to examine potential steric restrictions around the central chromatin axis. Tomograms were also observed and photographed on a varifocal (vibrating) mirror to illustrate the utility of a rapid user-interactive 3-D display.

Three-dimensional distribution of mineral in bone at a resolution of 15 micron determined by x-ray microtomography.

The application of x-ray microtomography (computerized axial tomography) to the histomorphometry of bone at a resolution of approximately 15 micron is described. Serial sections (obtained without physically cutting the sample) separated by 25 micron were made from an 0.8 mm square rod of human femoral bone. These showed several haversian canals and changes in mineralization within individual sections. The results obtained are quantitative in terms of the x-ray linear absorption coefficient, which is closely related to the degree of mineralization. Thus x-ray microtomography has the potential for the nondestructive determination of the fractional bone volume, with the ability to select the degree of mineralization of the bone used to determine the fraction.

Electron microscope tomography: transcription in three dimensions.

Three-dimensional reconstruction of an asymmetric biological ultrastructure has been achieved by tomographic analysis of electron micrographs of sections tilted on a goniometer specimen stage. Aligned micrographs could be displayed as red-green three-dimensional movies. The techniques have been applied to portions of in situ transcription units of a Balbiani ring in the polytene chromosomes of the midge Chironomus tentans. Current data suggest a DNA compaction of about 8 to 1 in a transcription unit. Nascent ribonucleoprotein granules display an imperfect sixfold helical arrangement around the chromatin axis.

X-ray microtomography.

A microscope system based on the principles of computerized axial tomography is described for determining the distribution of the X-ray absorption coefficient in a slice from a solid object without cutting sections. An application is given to determining the distribution at a resolution of about 15 micrometer through a shell of about 0.5 mm diameter.

Three-dimensional reconstruction from images of tilted specimens: the paramyosin filament.

The three-dimensional structure of the paramyosin filament has been reconstructed from images obtained when the specimen is tilted at different angles about its long axis. The micrographs were correlated spatially using gold marker beads and digitized. The different views contained in each micrograph were combined using a computational technique involving Fourier transforms and the resulting three-dimensional images are presented as various sets of serial sections. The results confirm that the Bear-Selby (1956) net can be seen at all levels in the specimen.