This paper presents a quantitative analysis of the structural parameters of gelatin gel networks derived from transmission electron micrographs. The network gel replicas were obtained by a quick-freezing, deep-etching and rotary replication method adapted to the study of fragile physical networks by Favard P. et al. (Biol. Cell 67 (1989) 201-207). Stereo electron micrographs were taken by tilting the replicas at increasing angles between – 35 to + 30○. A 3D reconstruction of the filamentous network was performed by stereoscopic and tomographic procedures. These procedures allowed us to measure the thicknesses of the replicas. The average length of filaments (triple helices) per unit volume and the average distance between filaments (mesh sizes of the networks) were derived for two polymer concentrations ($c=2~\%$ and $c=10~\%$). The results are in full agreement with optical rotation and small-angle neutron scattering measurements
SUMMARY Techniques for the preparation and investigation of thick biological sections in the high voltage electron microscope (HVEM) have been studied using the Toulouse 1.5 and 3 MV microscopes. Sections several micrometres thick can be made using conventional ultra‐microtomes, but, in order to know their actual thickness, it is necessary to resection them perpendicularly to their surface after embedding them in Araldite. When successive thick sections are made with glass knives, damage may be observed on their upper surface. This can be avoided by alternating several thin sections between each thick section. Such damage is further reduced if thick sections are made using diamond knives. The maximum thickness that can be observed at a voltage of 2.5 MV is not limited by the penetrating power of the beam but by the damage suffered by irradiated Araldite. The limit is approximately 10 μm. Below 10 μm a general uniform loss in thickness is observed in irradiated areas; above 10 μm, that loss in thickness is irregular and craters are formed under the beam. Contrast can be obtained by staining tissues before or after embedding them in Araldite. The cellular structures can be uniformly stained using uranium or lead salts, or selected parts of them can be stained using colloidal tracers, Gomori's lead method, osmium or silver impregnations. The latter techniques are the more convenient as far as sections several micrometres thick are concerned. The resolving power depends on section thickness and accelerating voltage. At 2.8 MV, by using selective staining techniques, sections several micrometres thick can be investigated with a sufficient resolving power, especially with regard to understanding the spatial organization. Stereoscopic viewing allows a better interpretation by removing the inconveniences resulting from overlapping structures, but the depth of field must then be considered. Its value, for a given resolving power, taking into account the focal length of the objective lens, depends on the objective aperture: by using a 5 μm aperture one can get a depth of field of 4 μm at a resolving power of 2.0 nm and 12 μm at a resolving power of 6.0 nm, the focal length of the Toulouse 3 MV electron microscope being 10 mm. At this voltage aberrations due to diffracting phenomena are not very disturbing.
Epithelial cells of frog urinary bladders fixed in different physiological states were examined by electron microscopy. It was shown: (1) that when bladders incubated with a hypotonic mucosal medium are water-permeabilized with oxytoxin, arginine-vasotocin, cyclic 3',5'-AMP and theophylline, this leads to a cellular swelling and the opening of intercellular channels; (2) that these effects are not observed when the transepithelial net water flow is suppressed by abolishing the external osmotic driving force; and (3) that modifications in the rate of active sodium transport do not change the morphological appearance of intercellular channels.These results are expecially discussed with respect to the localization to the intracellular site of the final effect of antidiuretic hormone on water permeability, and to the role of intercellular channels in the transepithelial transfer of water and sodium.
A microcomputer reconstruction technique has been developed in order to permit a larger exploitation of stereomicroscopy. The microcomputer facility consists of a digitizing tablet, a microcomputer, a graphics terminal, a graphics plotter and a printer. The technique has been applied to the study of HVEM stereopairs, performed by recording two images of the same area of a specimen (thick section of araldite-embedded leech ganglion neurons), tilted relative to the beam axis through an angle 0/20 degrees. Coordinates of N conjugate points of interest, expressed in a common reference system were obtained with the help of a digitizing tablet and the misorientation between the two images was determined by a method based on a least square technique. New projections of the object on different planes are provided by the microcomputer facility. Also the microcomputer method permits to obtain new stereopairs drawings, in various orientations and slices from a three-dimensional reconstruction of the object oriented in any direction in space. The method permits to obtain computed anaglyph drawings, printed here, which are stereoviews of the same object.
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