Energy Filtering Imaging Of Thick Biological Specimens With In-Column "Omega"-Filter Microscopes.

Preliminary results were obtained from thick sections of biological specimens using 200 keV electron microscopes with an in-column “Omega” energy filter. Images of selectively stained specimens prepared by osmium impregnation of frog spinal ganglia, with thickness ranging from 0.5 μm to 2.5 μm, show drastic improvement in image quality with the use of the energy filter.Two instruments with Omega filters were used. One was a non-commercial research prototype at JEOL Ltd. equipped with a LaB6 gun and the other was a commercial instrument (JEM-2010FEF) with a field-emission gun, installed at Kyushu University. We found that, although the field-emission gun offered a brightness 2 to 3 orders of magnitude higher, the LaB6 gun was more suitable for studying thick specimens, primarily due to the higher beam current of the LaB6 gun.

Evidence of a Tubular System for Transendothelial Transport in Arterial Capillaries of the Rete Mirabile

The arterial endothelial cells of the rete capillaries of the eel were examined by transmission electron microscopy on thin sections, on freeze-fracture replicas, by scanning electron microscopy, after cytochemical osmium impregnation and perfusion with peroxidase. The study revealed the existence of membrane-bound tubules and vesicles that open at both the luminal and abluminal poles of the cell and at the level of the intercellular space. The tubules are straight or present successive dilations and constrictions. They branch in various directions and intrude deeply into the cell cytoplasm, forming a complex tubular network within the cell. Immunocytochemical techniques were applied on immersion-fixed tissues and on perfusion of the capillaries with albumin and insulin. These demonstrated that the tubular–vesicular system is involved in the transport of circulating proteins. Furthermore, protein A–gold immunocytochemistry has revealed the association of actin with the membranes of this system. On the basis of these results, we suggest that the transendothelial transport of serum proteins takes place by a transcytotic process through a membrane-bound tubular–vesicular system and is equivalent to the large pore system presumed from functional studies.

A new method based on cobalt for histochemical and cytochemical demonstration of glucose-6-phosphatase activity.

We describe a novel technique for the histochemical and cytochemical demonstration of glucose-6-phosphatase activity. In this method, lead is replaced by cobalt. After activity of glucose-6-phosphatase, cobalt phosphate Co3(PO4)2 is formed, and in the presence of ammonium sulfide (NH4)2S, the precipitate is transformed into a sulfide that fixes osmium and provides good electron density. Glucose-6-phosphatase activity was determined mostly in rat kidney cells, but controls were also performed in liver cells. A strong reaction was seen in proximal tubule cells, but the reaction was weak in distal convoluted tubule cells. This technique showed the same endoplasmic reticulum (ER) organization in proximal and distal nephron as that seen with the osmium impregnation technique. In collecting tubules, intercalated cells had irregular reactivity, while principal cells had none. Our results indicate that the cobalt technique is valid, reliable, and sensitive enough to detect low glucose-6-phosphatase activity. Moreover, the technique can be used with 1-mm-thick specimens and obviates the need for use of frozen tissue sections.

Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study.

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.