Electron-dense Substance Research Articles

Ultrastructural Characteristics of Alginate-Induced Reddish Purple Stained Cells (ARPC) in Mouse Peritoneal Cavity

He, W., Fujiki, K. and Fujihara, N. 2001. Ultrastructural characteristics of alginate-induced reddish purple stained cells (ARPC) in mouse peritoneal cavity. J. Appl. Anim. Res., 20: 15–26. To characterize microscopic features of the alginate-induced reddish purple-stained cells (ARPC) using electron microscope, one ml of sodium alginate solution (10 mg/ml) was injected into mice intraperitoneally. Mice were killed after the injection at various time intervals to collect the peritoneal fluid. The cells contained in the fluid were collected by centrifugation and subjected to cytological examinations. As a result, electron microscopy revealed that the vacuole-like organelles (VLO) contained very small amount of electron-dense substance. The substance was stained reddish purple with Wright solution. ELISA using anti-alginate monoclonal antibody and uronic acid determination indicated that the substance is not phagocytosed sodium alginate but ARPC-derived cellular materials. It is concluded that the ARPC is shown to be obviously cellular organelles.

Electron microscopic cytochemical studies of anionic sites in the rat spleen.

The distribution patterns of the intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of endothelial cells (ECs) in blood vessels and reticular cells (RCs) in the splenic cord of the rat spleen were studied by an electron microscopic cytochemical method using polyethyleneimine (PEI) as a cationic probe. Spleens from adult male rats were perfusion-fixed with 0.5% glutaraldehyde -4% paraformaldehyde containing 0.05% cetylpyridinium chloride and then perfused with 0.5% PEI at pH 7.4. On the free surfaces (glycocalyx of the plasma membrane) of the ECs examined, distinct PEI-positive reactions were observed in blood vessels, such as trabecular arteries, central arteries, arterial capillaries, pulp veins and trabecular veins. These PEI-positive electron-dense substances in the trabecular arteries, central arteries, and trabecular veins took the shape of a band of 170-250 nm in thickness. On the other hand, the corresponding ultrastructure of the ECs lining the splenic sinuses and the RCs in the splenic cord showed exceedingly weak PEI reactions. The PEI-reactive deposits were significantly thinner than those in the above blood vessels. As the thickness of the electron-dense substances can be related to the density of the negative charge, these results suggest that there is a high intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of ECs in blood vessels where blood cells and plasma pass into the red pulp or are discharged from the red pulp. In contrast, the splenic sinuses and RCs, which are the main components of the red pulp, contain weakly negative-charged sites. This may contribute to the microcirculation of the splenic blood vessels and elucidate the possible physiological functions of the spleen, such as blood storage.

The morphology of Protoopalina pomacantha, n. sp., symbiont in the rectum of the Angelfishes, Pomacanthus zonipectus and Holacanthus passer. A light, scanning electron and transmission electron microscopic study

Protoopalina pomacantha n. sp. were discovered with fecal debris from the large hind gut chamber (rectum) of the Angelfishes, Pomacanthus zonipectus, and Holacanthus passer, collected from the Sea of Cortez near La Gaviota Island, Mexico. To describe this species, specimens from each host were studied with both light and electron microscopy. It is generally similar to other fish inhabiting protoopalines. The body is curved, contains a small anterior falx, and a pointed posterior end-barren of flagella. It has desmose fibrils interconnecting kinetosomes and electron dense substance within kinetosomes. The form of it’s somatic kinetosomal desmose is similar to at least one species of the genus, Protoopalina, but differs from several. The falx kinetosomal and desmose ultrastructure is similar to several opalinids. This is judged to be a new species based on its host, components within the kinetosomal lumen, and desmose structures. Bacterial ectosymbionts are often found attached, in shallow pits, within a kinety and between flagella.

The digestive cells of the hepatopancreas in Aplysia depilans (Mollusca, Opisthobranchia): ultrastructural and cytochemical study

Digestive cells are the most abundant cell type in the digestive diverticula of Aplysia depilans. These are tall columnar or club shaped cells, covered with microvilli on their apical surface. A large number of endocytic vesicles containing electron-dense substances can be found in the apical zone, but the presence of many heterolysosomes of large diameter is the main feature of these cells. Glycogen particles and some lipid droplets were also observed. Peroxisomes with a circular or oval profile were common, but crystalline nucleoids were not detected in them, although a dense spot in the matrix was observed in a few cases. These organelles were strongly stained after cytochemical detection of catalase activity. The Golgi stacks are formed by 4 or 5 cisternae, with dilated zones containing electron dense material. Arylsulphatase activity was detected in the Golgi stacks and also in lysosomes. Cells almost entirely occupied by a very large vacuole containing a residual dense mass seem to be digestive cells in advanced stages of maturation. The observation of semithin and ultrathin sections indicates that these very large vacuoles are the result of a fusion among the smaller lysosomes. Some images suggest that the content of these large vacuoles is extruded into the lumen of the digestive diverticula.

Cellular analyses of sea urchin metamorphosis

Larvae of marine invertebrates undergo metamorphosis in response to environmental cues (Chia & Burke, 1978). In sea urchins, free fatty acids (Kitamura et al., 1993), dibromomethane (Taniguchi et al., 1994), pheromonal peptides (Burke, 1984) and L-glutamine (Yazaki & Harashima, 1994; Yazaki, 1995) have been known as metamorphosis-inducing substances. The mechanisms by which cells respond to these cues and how the larval tissues are absorbed have not been clear, however. In the present study, we used L-glutamine (Gln) and a natural cue, green algae (Ulvella sp.), to induce metamorphosis of Hemicentrotus pulcherrimus and Anthocidaris crassispina, and investigated the intracellular changes during metamorphosis.After being subjected to 10−5–10−3 M Gln for 10–24 h, larvae cease swimming, settle, begin to retract their larval arms, extrude the primary podia and finally evert their echinus rudiment (ER). In H. pulcherrimus, larvae retracted their arms from 6 h to 24 h after the start of Gln treatment and then everted the ER. A. crassispina larvae underwent similar processes to those of H. pulcherrimus. The larval surface is composed of squamous epithelium and columnar epithelium. The epithelium of the ciliary bands or epaulets is columnar.In the squamous epithelium, the nuclear chromatin in the larval arms and body, and in the oesophagus, markedly condensed after treatment with Gln for 24 h. Electron microscopy revealed swelling of both nuclei and mitochondria, while their membranes seemed to be intact. In the cytoplasm, lipid-like structures and electron-dense substances appeared. A further 24 h after Gln treatment, the chromatin condensation had progressed. Most nuclei in which chromatin had condensed were positive to the TUNEL assay, which detects DNA fragmentation. These results suggest that cell death in the squamous epithelium is apoptotic rather than necrotic.

Neuromusculature of the ovijector of ascaris suum (Ascaroidea, nematoda): an ultrastructural and immunocytochemical study.

This study used electron microscopy and confocal scanning laser microscopy interfaced with cytochemistry to study neuromuscular interrelationships in the ovijector of Ascaris suum. An extensive nerve plexus with both FaRPergic and non-FaRPergic components extends over the outer surface of the ovijector. The non-FaRPergic component is derived from nerve branches of the ventral nerve cord, whereas the FaRPergic component emanates from two large FMRFamide-immunoreactive neurons. In the vagina vera, most myofibrils are circular in orientation and a number of them divide and run for short distances in longitudinal and diagonal directions, their myofilaments are also orientated in a variety of directions. Parallel nerve fibres run in tracts along the length of the vagina vera with branches that penetrate the muscle layers. The vagina uteri possesses a thicker hypodermis than that of the vagina vera. It appears rich in secretory and phagocytic vesicles and the luminal side is invested with an electron-dense substance. The musculature of the vagina uteri is less well developed than that of the vagina vera, being restricted to circular myofibrils, with an apparent diagonal arrangement of myofilaments. Also, the innervation is less extensive in the vagina uteri with many fibres returning to the vagina vera to rejoin the nerve net and others continuing into the uteri.

Fine structure of dental epithelial cells and the enameloid during the enameloid formation stages in an elasmobranch, Heterodontus japonicus.

The structural features of the dental epithelial cells and the enameloid in tooth germs of the Japanese Port Jackson shark, Heterodontus japonicus, in the stages of enameloid formation, were investigated by light and transmission electron microscopy. At the enameloid matrix-formation stage, tall columnar inner dental epithelial cells contained large numbers of glycogen particles. At the enameloid mineralization stage, when many sharply outlined crystals appeared throughout the enameloid, the inner dental epithelial cells exhibited well-developed Golgi apparatuses and many mitochondria in the proximal cytoplasm, and abundant vesicles and vacuoles in the distal cytoplasm. Marked interdigitations of the lateral membrane were visible in the inner dental epithelial cells. The outer dental epithelial cells contained many mitochondria, lysosomal bodies, vesicles and microtubules, and the capillaries usually approached the outer dental epithelial cells. At the enameloid maturation stage, large numbers of crystals occupied the enameloid, and most of the organic matrix had disappeared from the enameloid area after demineralization. The organelles in the inner and outer dental epithelial cells decreased in number, but there were still widely distributed Golgi apparatuses, abundant intermediate filaments and granules containing an electron-dense substance in the inner dental epithelial cells. It is probable that the dental epithelial cells are involved in the removal of organic matrix from the enameloid and in the process of mineralization at the later stages of enameloid formation, i.e., the mineralization and the maturation stages.

The ultrastructure of some endogenous stages of the coccidian Eimeria boveroi Carini & Pinto, 1926 in the gut epithelial cells of the gecko Hemidactylus mabouia from Brazil.

The ultrastructure of intracytoplasmic meronts and macrogamonts of Eimeria s.l. boveroi in the small intestine of the gekkonid lizard Hemidactylus mabouia from Belem, Para north Brazil is described. Young meronts, and some of the fully grown macrogamonts, are coated with a glycocalyx which, in cross-section, has the appearance of a series of fine tubules. The wall of the parasitophorous vacuole (PV) is a single membrane, lined on the host-cytoplasm side with an endoplasmic reticulum (ER) which sometimes expands into large cisternae filled with electron-lucent, globular material. The membranal edges of the ER, canaliculi and cisternae have regularly spaced indentations filled with an electron-dense substance. Vesicular mitochondria are present in addition to those of the conventional type. Macrogamonts develop the characteristic type 1 and type 2 wall-forming bodies, and in some zygotes the cisternae containing the latter are grossly expanded. Both types of wall-forming bodies persist in young oocysts that have a distinct oocyst wall.

Effects of Milsana and Benzothiadiazole on the Ultrastructure of Powdery Mildew Haustoria on Cucumber

ABSTRACT Disease assessments and cytological investigations provided valuable information on the modes of action and efficacies of two prophylactic compounds, Milsana and benzothiadiazole (BTH), against powdery mildew development on long English cucumber. Milsana application significantly reduced disease incidence relative to inoculated controls through induction of localized resistance. Microscopic observations showed most haustoria had collapsed in the localized Milsana treatment and were encapsulated by an amorphous material impregnated by electron-opaque substances. The rapidity of haustorial collapse (within 4 days of treatment application) together with the encasement by electron-dense substances stained blue by toluidine blue O suggest that phenolics are possibly involved in the Milsana defense response. Cytochemical labeling of chitin with a wheat germ agglutinin/ovomucoid-gold complex showed that complete cellular disorganization of the fungus had occurred without disturbance to chitin in the walls of mycelia and haustoria. This may indicate that chitinolytic activity is not important in the Milsana-activated defense response. Application of high doses of BTH induced occasional cell wall thickening and accumulation of a compound that stained purple by toluidine blue O, but the defense response was weak, sporadic, and insufficient to reduce powdery mildew infection on cucumber. Responses to BTH could not be differentiated in terms of timing of the initial application or systemicity.

Transformation of trans-Golgi Network During the Cell Cycle in a Green Alga, Botryococcus braunii

-Golgi network (TGN), and the changes in its structure and behavior throughout the cell cycle of a unicellular green alga, Botryococcus braunii, were examined with deep-etching replicas and in cryo-fixed/freeze-substituted specimens. In interphase cells, the TGN consisted of a hemispherically shaped cisterna (TGN-cisterna) with regularly distributed pores on the surface and a tubular network (TGN-tubules) with clathrin-coated vesicles. The TGNs changed their structure drastically throughout the cell cycle. The TGN-cisterna disappeared from the beginning of nuclear division to the completion of the cell wall, in contrast that TGN-tubules with the clathrin-coated vesicles were always observed. The TGN-tubules produced at least five other kinds of vesicles depending on the stage of the cell cycle: 200-nm vesicles with fibrillar substances and multivesicular bodies in interphase, 180–240 nm vesicles during cell division, and 400–450 nm vesicles containing fibrils and small masses of electron-dense substances, and 200-nm vesicles containing electron-dense spherical substances just after cell division. During cell wall formation, TGN-tubules were small and had only a few clathrin-coated vesicles. After cell wall formation, TGN-tubules grew and a TGN-cisterna with pores appeared again.

Histological observations of latent infection and tissue colonization by Diaporthe toxica in resistant and susceptible narrow-leafed lupins

Latent infection and tissue colonization by Diaporthe toxica was examined by light, scanning, and transmission electron microscopy in stems, leaves, and petioles of resistant and susceptible narrow-leafed lupins (Lupinus angustifolius). Resistance was observed during the latent phase of the disease as an incompatible reaction between the host and pathogen that appeared to occur after penetration of the cuticle. Conidia were attached firmly to the cuticle by an extracellular substance presumably exuded from the conidia. Conidia penetrated the cuticle directly via an infection peg and formed subcuticular coralloid hyphae. The frequency of subcuticular coralloid hyphae was similar on stems, leaves, and petioles of each line. At 14 days after inoculation, resistant plants had a high frequency of small coralloid hyphae (10-80 µm length). The epidermal cells beneath these small coralloid hyphae appeared necrotic and collapsed with accumulation of polyphenolics and electron-dense substances and a loss of internal organisation in the cytoplasm. Necrosis was occasionally observed in small coralloid hyphae as well. Susceptible plants had a high frequency of large coralloid hyphae (80-400 µm length) in which intrahyphal hyphae were observed, and host epidermal cells beneath large coralloid hyphae appeared normal. Colonization of tissues below the cuticle began immediately after excision of stems from susceptible plants, but was delayed in resistant plants. At 8 days after excision, hyphae had invaded all stem tissues and initiated the formation of pycnidia in susceptible plants, but few hyphae were observed in stems of resistant plants.Key words: Diaporthe toxica, coralloid hyphae, Lupinus angustifolius, resistance.

Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and cytochemistry of the host response

The potential of Bacillus pumilus (PGPR strain SE 34), either alone or in combination with chitosan, for inducing defense reactions in tomato (Lycopersicon esculentum Mill.) plants inoculated with the vascular fungus, Fusarium oxysporum f. sp. radicis-lycopersici, was studied by light and transmission electron microscopy and further investigated by gold cytochemistry. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm signal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by biotic agents. Ultrastructural investigations of the infected root tissues from water-treated (control) plants showed a rapid colonization of all tissues including the vascular stele. In root tissues from bacterized tomato plants grown in the absence of chitosan, the limited fungal development coincided with marked changes in the host physiology. The main facets of the altered host metabolism concerned the induction of a structural response at sites of fungal entry and the abnormal accumulation of electron-dense substances in the colonized areas. A substantial increase in the extent and magnitude of the cellular changes induced by B. pumilus was observed when chitosan was supplied to bacterized tomato plants. These changes were characterized by a considerable enlargement of the callose-enriched wall appositions deposited onto the inner cell wall surface in the epidermis and the outer cortex. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin component in Fusarium cells colonizing bacterized tomato roots was not substantially altered. One of the most-typical fungal cell reactions, observed only when bacterized tomato plants were grown in the presence of chitosan, was the formation of abnormal chitin-enriched deposits between the retracted plasma membrane and the cell wall. Results of the present study provide the first evidence that combination of biocontrol approaches is a promising step towards elaborating integrated pest management programmes.

Lung lesions in human leptospirosis: microscopic, immunohistochemical, and ultrastructural features related to thrombocytopenia.

Lung fragments from 12 patients were collected immediately after death and studied by light and electron microscopy and by immunohistochemistry to describe the main morphologic and ultrastructural aspects of the lung and platelets in leptospirosis (Weil's syndrome), to search for the possibility of disseminated intravascular coagulation (DIC), and to assess the relationship between endothelial lesions and local platelet aggregation and the leptospiral antigen distribution, as well as its relationship with the intensity of the lesions. The immunohistochemical results for fibrin aggregates were positive in the lumen and/or on the vascular endothelium in nine cases and on the alveolar surface in seven cases, leading to the diagnosis of the adult respiratory distress syndrome in these seven cases. Test results for leptospiral antigen by immunohistochemistry were positive in eight cases with no direct relationship between antigen deposits in the pulmonary vascular endothelium and intensity of the lesions. The ultrastructural findings were uniform and constant. Capillary lesions were characterized by swelling of endothelial cells, an increase in pinocytotic vesicles, and giant dense bodies in the cytoplasm of these cells. No necrosis, rupture, nor exposed subendothelial collagen was observed outside the hemorrhagic areas, and the intercellular junctions were preserved. The lung involvement in severe human leptospirosis presents as hemorrhagic pneumopathy with septal capillary lesions that are the usual cause of death. The thrombocytopenia that was verified in 11 of 12 patients in our study seems to bear no relationship to DIC and seems to be determined by activation, adhesion, and aggregation of platelets to the stimulated vascular endothelium, with an amorphous electron-dense substance between the endothelial cells and the adherent platelets in places where the subendothelial collagen was not exposed.

Ultrastructural aspects of the intact titanium implant-bone interface from undecalcified ultrathin sections

An osseointegrated oral implant with surrounding bone was used for electron microscopical analyses of the implant-bone interface. The bulk metal was removed by sawing and grinding techniques, leaving only the plasma-sprayed titanium coating anchored in mineralized bone. Ultrathin sections were realized from these reduced interface areas and underwent ultrastructural and crystallographic assessments. The microscopical observations showed that ultramicrotomy was suitable for producing such interface sections. Two different, concomitant, interfacial structures were noticed. On the one hand it was possible to observe bone crystals directly apposed on the implant surface; on the other, a granular electron-dense substance was interposed between the plasma-sprayed coating and the bone. The applied technical approach allows one to study the osseointegration process, at high resolution levels, of intact interfaces from complete osseointegrated implants.

Postpollination-prezygotic ovular secretions into the micropylar canal inPseudotsuga menziesii (Pinaceae)

Ovular morphology was examined ultrastructurally inPseudotsuga menziesii to determine the effects of the ovule on pollen development. Vesicles containing lipid-like substances traverse cell walls of the inner epidemis of the integument and release their contents at the integument surface to form the integumentary membrane. A major aqueous secretion from the integument into the micropylar canal is proposed to occur by the movement of the integumentary membrane and its invaginations towards the center of the micropylar canal. The cellular degeneration of the nucellar apex results from the breakdown of vacuoles. After this degeneration, electron-dense substances move from the prothallial cells of the female gametophyte towards the nucellus, and many morphological changes in the nucellus, prothallial cells, and micropylar canal take place simultaneously. We interpret these changes to result from another major secretion from the prothallial cells. Egg cytoplasm appears to disorganize for a short time. Simultaneously, substantial amounts of electron dense-substances in the prothallial cells and lipid-like substances in surface cell walls of the female gametophyte move towards the nucellus as components of the third major secretion.

Pollination Drop and Microdrop Secretions in Cedrus

In Cedrus pollen adheres by microdrops to the apical portion of the integument. The microdrops originate from dictyosome vesicles in cells of the integument apex. Electron-dense substances are present in these dictyosome vesicles and accumulate between the surface cell walls and plasma membrane. The substances traverse the walls and form microdrops. The cuticle layer on the surface cell walls is less developed and does not serve as an obstacle to the passage. During pollination drop secretion, nucellar cells show characteristics such as undulation of the plasma membrane, numerous dictyosomes, and a prominent amount of endoplasmic reticulum. The cuticle layer is absent from the nucellar apex. After the secretion, cell walls of the nucellus and chalaza become transparent. We describe ultrastructural changes associated with the winter dormancy of the ovule.

Axonal dystrophy in monkey spinal ganglia: Involvement of the initial segment of axons

Examination of spinal ganglia of ageing and old Japanese monkeys, Macaca fuscata, by light microscopy and electron microscopy revealed unidentified pathology of the axon initial segment. Although the incidence of this change is infrequent in any one histological section of ganglion, its morphology is quite remarkable. The change is characterized by a progressive swelling of the unmyelinated portion of axon followed by continuous involvement of the first myelin segment. Neurofilaments proliferate in rather randomly oriented bundles within the affected axons accompanied with a considerable amount of mitochondria. The axons, originally 3–5 um in diameter, thus become nodularly varicose and become as thick as 20 um or more in diameter. The second stage of the axonal pathology is characterized by massive dense accumulation of tiny vesicular granules which infiltrates and/or replaces the neurofilament mass, almost fills the whole axon and the affected axons could result in a thickness of 70 um or more in diameter (i.e. larger than the mother perikaryon itself). The third stage of the axonal pathology is characterized by a catabolic transformation of those dense massive aggregates of the vesicular granules into irregular patches of amorphous electron dense substance. Smooth endoplasmic reticulum may make a focus of proliferation within the affected axon, but its development is relatively poor and restricted. It is surprising that the mother perikaryon of the affected axon initial segment remains as a rule quite normal in appearance. It is also remarkable that the part of the axon which is distal to the swelling remains viable, apparently for a long time. The significance of this axonal pathology is discussed in relation to ageing and diabetic axonal dystrophy in the sympathetic ganglia, and to ageing axonal dystrophy in the gracile nucleus.

Rat intestinal galactoside-binding lectin L-36 functions as a structural protein in the superficial squamous cells of the esophageal epithelium.

Using an affinity purified antibody raised against the RI-H fragment of rat intestinal lectin L-36, the latter protein has been identified within the esophageal epithelium by means of ultracryotomy followed by immunogold labeling. The epithelium consists of 4 morphologically distinct cell-types, namely, the basal, spiny, granular and squamous cells, and each of these exhibits a different immunolabeling pattern. The basal cells form a layer on the basal lamina, and in these a diffuse cytoplasmic staining is observed. This basal cell layer is overlaid by spiny cells that extend many cell processes into wide intercellular spaces. In these cells, immunogold particles are found only on small granular inclusions consisting of an electron-lucent homogeneous substance. The granular cells form a third layer over the spiny cells, and are characterized by a number of large granular inclusions with an electron-dense core rimmed by a less electron-dense substance. Immunogold labeling is found on these granules, both on the core and peripheral region. Squamous cell-types constitute the most superficial layer of the epithelium. They are without granular inclusions, and immunogold labeling is confined to the cytoplasmic surface of the thickened plasma membrane. These findings suggest that L-36 is produced in the basal cells as free cytosolic protein, then becomes progressively aggregated into the granular inclusions of the spiny and granular cells, and is eventually transferred onto the cytoplasmic surface of the squamous cell plasma membrane where it may interact with complementary glycoconjugate(s) located at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

Lichen sclerosus et atrophicus. A histological, immunohistochemical and electron microscopic study.

We studied three cases of genital lichen sclerosus et atrophicus (LSA) using histological, immunohistochemical and ultrastructural techniques to elucidate the characteristics of the collagen fibres, the elastic fibres and the interfilamentous matrix in the upper dermal homogeneous zone. In the early stages of LSA, the homogeneous zone caused elaunin fibres to push downwards and partially disappear, the collagen fibres were presumably phagocytosed by fibroblasts, and there were peculiar cells containing numerous vacuoles in the cytoplasm. In the advanced stages of LSA, the homogeneous zones showed newly formed collagen fibres and amorphous, dispersed, medium electron-dense substances that were probably composed of elastin or elastin-like substances and microfilaments originating from the collagen fibres. Immunohistochemically, there were numerous anti-aortic alpha-elastin-positive substances, but there was no positivity for anti-amyloid P component. These findings suggest that in advanced LSA a hybrid substance (elastocollagenous mass) between collagen fibres and elastic fibres may be formed in the homogeneous zone.

Electron-dense Regions in Dividing Mitochondria of a Green Alga, Scenedesmus acutus

Abstract The dividing and the constricted regions of mitochondria in a green alga, Scenedesmus acutus , were studied by electron microscopy. Electron-dense substances were always visible in these specific regions of mitochondria that had been fixed by freeze-substitution. Thin fibres, which seemed to surround the constricted regions, were also seen. The possibility that the electron-dense regions constitute part of the dividing apparatus of the mitochondria is discussed.