Rhabdomeric membrane and smooth endoplasmic reticulum in photoreceptors of Manduca sexta: modulations associated with the diurnal light/dark cycle and effects of chromophore deprivation

Summary Aberrations of photoreceptor ultrastructure resulting from carotenoid/retinoid (vitamin A) deprivation were studied in the retina of Manduca sexta. The syndrome of chromophore deficiency included hypertrophy of smooth endoplasmic reticulum, variable dilation of rhabdomeric microvilli, the insertion of endomembrane fingers into such enlarged microvilli, and the formation of rhabdomeric vacuoles, intracellular compartments containing microvilli similar to those of the rhabdomere. Retinas were processed either with conventional procedures employing preliminary aldehyde fixation followed by heavy metal postfixation, or by fixation and incubation in unbuffered OsO₄. The latter method deposits osmium throughout the endomembrane system, within the rhabdomeric vacuoles, and in the extracellular space of the rhabdom. However, the intravillous fingers were rarely impregnated with osmium, despite their continuity with densely stained cisternae of the smooth endoplasmic reticulum. We suggest that the insertion of endomembrane fingers into dilated microvilli results from a cytoskeleton-mediated link between cisternae of the smooth endoplasmic reticulum and the rhabdomeric membrane, an association that may be important in the turnover of photoreceptor membrane. We interpret endomembrane hypertrophy and development of rhabdomeric vacuoles as symptoms of disturbance in the pathway leading to the assembly of the rhabdomere resulting from reduced synthesis of visual pigment.     

Über fixationsbedingte Unterschiede in der elektronenmikroskopischen Morphologie der Zelltypen im Inselapparat des Frosches Rana ridibunda

Zusammenfassung Langerhanssche Inseln des Frosches Rana ridibunda wurden nach Osmiumtetroxyd-, Glutaraldehyd- und Acroleinfixierung elektronenmikroskopisch untersucht. Nach ausschließlich Aldehydfixierung und Nichtanwendung einer Kontrastierungsmethode fehlt den Sekretgranula der Inselzellen ihr bekannter positiver Kontrast; sie können sogar im negativen Kontrast erscheinen. B-Zellen besitzen runde und nadeiförmige Sekretgranula neben anderen Zelleinschlüssen (Lipidtropfen, lysosomenartige Einschlüsse, Vakuolen, glykogenartiges Material) und zeigen meist eine bipolare Zytoplasmaorganisation. Golgiapparat und Ergastoplasma sind relativ spärlich entwickelt. Während die B-Zellmorphologie keine starke Abhängigkeit von der Art der Fixierung zeigte, erwies sich das Bild der Typ IIundTyp HI-Zellen als stark fixationsabhängig. So scheinen chromophobe Zellen, d.h. Zellen, die leere Bläschen an Stelle der Sekretgranula enthalten, bei Osmiumfixierung aus Typ IIZellen und bei Acroleinfixierung aus Typ III-Zellen zu entstehen. Chromophobe Zellen wurden nach Glutaraldehydfixierung niemals beobachtet. Die bläschenreichen Zellen erscheinen den D-Zellen verschiedener Autoren verwandt zu sein. Das Bild dieser Elemente wird als Fixationsartefakt betrachtet. Ein eigentlicher chromophober Zelltyp scheint demnach in den Langerhansschen Inseln des Frosches nicht zu existieren. Die hellen Zellen der Lichtmikroskopie rekrutieren sich demnach aus verschiedenen Quellen, zu denen granulaarme Formen aller 3 Zelltypen und schlecht erhaltene granulareiche Formen der Typen II und III gehören.

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Summary In a study on the fine structure of the endocrine pancreas in Rana ridibunda, osmium tetroxide, glutaraldehyde, and acrolein have been used as fixatives. After aldehyde fixation alone, without application of any further contrasting procedure, the secretory granules of the islet cells lack their known positive contrast and may show up even in negative contrast. B cells possess round and needle-like secretory granules together with other inclusions (lipid, lysosome-like, vesicular, glycogen-like) and mostly display a bipolar organisation. B cell Golgi apparatus and ergastoplasm are relatively sparse. Whilst B cell structures do not show much variation with the fixative employed, those of type II and type III cells do. Thus, chromophobe cells, i.e. cells containing empty vacuoles instead of secretory granules, seem to arise from type II cells with osmium tetroxide fixation and from type III cells with acrolein fixation. Chromophobe cells were never observed after glutaraldehyde fixation. They are likened to D cells of authors employing osmium fixation and are considered to be artifacts due to fixation. No chromophobe cell proper, therefore, seems to exist in the frog pancreatic islet and the clear cells of light microscopy should stem from several sources including agranular stages of the types B, II, and III and badly preserved granular stages of types II and III.

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Durchgeführt mit dankenswerter Unterstützung durch den Schweizerischen Nationalfonds.

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Zusatz bei der Korrektur. Forssmann, W. G., G. Siegrist, L. Orci, L. Girardier, R. Pictet und Ch. Roullier berichteten [J. Microscopie 6, 279–304 (1967)] über elektronenmikroskopische Beobachtungen an verschiedenen Organen nach Perfusionsfixierung. Die in der vorliegenden Studie nach Immersionsfixierung erhobenen Befunde mit solchen nach Perfusionsfixierung zu erhebenden zu vergleichen, erscheint uns wesentlich.     

The classic Golgi apparatus and vacuoles

The dense vacuoles, considered to be the classic Golgi apparatus in the root meristem ofFagopyrum, were studied by the following methods: 1. Impregnation methods for the demonstration of the Golgi apparatus, 2. cytochemical methods, 3. electron microscopic methods in the light microscope and 4. the electron microscope. A comparison was made with the classic Golgi apparatus in animal cells in the light and electron microscope. Dense vacuoles inFagopyrum and also evidently in other plants, were taken for the classic Golgi apparatus on account of their morphological similarity to the Golgi apparatus in animal cells on impregnation with silver and osmium and their staining preperties with lipoid methods. Dense vacuoles differ from the classic Golgi apparatus in other chemical properties, such as content of phenol substances, etc. No formations were found in animal cells which were similar to dense vacuoles on investigating by electron microscopy. In the electron microscope dense vacuoles have the appearance of derivatives of the normal light vacuoles known in plant cells. They therefore belong to vacuome of plant cell and cannot be analogous to the classic Golgi apparatus in animal cells. Thus the use of the term Golgi apparatus for dense vacuoles is not well founded. A comparison was made of fixation and impregnation used in the light microscope with fixation in the electron microscope. After fixation with permanganate, dense vacuoles have the same shape as after impregnation. After fixation with permanganate, they stain an intense black in the same way as after impregnation with silver and osmium. The form of the vacuoles is dependent on the fixation used. The comparison was made in the light microscope.     

Chorioallantoic placenta formation in the rat: II. Angiogenesis and maternal blood circulation in the mesometrial region of the implantation chamber prior to placenta formation.

Rat gestation sites were examined on days 7 through 9 of pregnancy by light microscopy and transmission and scanning electron microscopy to determine the extent of vascular modifications in the vicinity of the mesometrial part of the implantation chamber (mesometrial chamber). At a later time, the mesometrial chamber is, in conjunction with the uterine lumen, the site of chorioallantoic placenta formation. On day 7, in the vicinity of the mesometrial chamber, vessels derived from a subepithelial capillary plexus and venules draining the plexus were dilating. By early day 8, this network of thin-walled dilated vessels (sinusoids) was further enlarged and consisted primarily of hypertrophied endothelial cells with indistinct basal laminas. Sinusoids were frequently close to the mesometrial chamber's luminal surface which was devoid of epithelial cells but was lined by decidual cell processes and extracellular matrix. By late day 8, cytoplasmic projections of endothelial cells extended between healthy-appearing decidual cells and out onto the mesometrial chamber's luminal surface, and endothelial cells were sometimes found on the luminal surface indicating that endothelial cells were migrating. The presence of maternal blood cells in the mesometrial chamber lumen suggested that there was continuity between the chamber and blood-vessel lumens. On day 9, the mesometrial chamber was completely lined with hypertrophied endothelial cells, and sinusoid lumens were clearly continuous with the lumen of the mesometrial chamber. Mesometrial sinusoids and possibly the mesometrial chamber lumen were continuous with vessels in vicinity of the uterine lumen that were fed by mesometrial arterial vessels. Clearing of the mesometrial chamber lumen during perfusion fixation via the maternal vasculature indicated the patency of this luminal space and its confluence with mesometrial arterial vessels and sinusoids. The conceptus occupied an antimesometrial position in the implantation chamber on days 7 through 9, and it was not in direct contact with uterine tissues in the vicinity of the mesometrial chamber. These observations suggest that angiogenesis, not trophoblast invasion or decidual cell death, plays a major role in the opening of maternal vessels into the mesometrial chamber lumen before the formation of the chorioallantoic placenta.     

The passage of macrophages and lymphocytes from the interstitium across the lymphatic endothelium of rat lacteals

Summary The passage of cells across the lymphatic endothelium of rat lacteals in both normal and non-pathological experimental conditions (fasting, lymphatic stasis) was studied by means of serial thin sections and three-dimensional models. Two different pathways of transendothelial migration were observed: (1) macrophages enter the lymphatic lumen via the cytoplasm of endothelial cells, without involvement of intercellular junctions, whereas (2) lymphocytes migrate through intraendothelial channels, dynamic structures organized by the lymphatic endothelium under physiological conditions.     

Combinatory action of VEGFR2 and MAP kinase pathways maintains endothelial-cell integrity

Blood vessels normally maintain stereotyped lumen diameters and their stable structures are crucial for vascular function. However, very little is known about the molecular mechanisms controlling the maintenance of vessel diameters and the integrity of endothelial cells. We investigated this issue in zebrafish embryos by a chemical genetics approach. Small molecule libraries were screened using live Tg(kdrl:GRCFP)(zn1) transgenic embryos in which endothelial cells are specifically labeled with GFP. By analyzing the effects of compounds on the morphology and function of embryonic blood vessels after lumen formation, PP1, a putative Src kinase inhibitor, was identified as capable of specifically reducing vascular lumen size by interrupting endothelial-cell integrity. The inhibitory effect is not due to Src or general VEGF signaling inhibition because another Src inhibitor and Src morpholino as well as several VEGFR inhibitors failed to produce a similar phenotype. After profiling a panel of 22 representative mammalian kinases and surveying published data, we selected a few possible new candidates. Combinational analysis of these candidate kinase inhibitors established that PP1 induced endothelial collapse by inhibiting both the VEGFR2 and MAP kinase pathways. More importantly, combinatory use of two clinically approved drugs Dasatinib and Sunitinib produced the same phenotype. This is the first study to elucidate the pathways controlling maintenance of endothelial integrity using a chemical genetics approach, indicating that endothelial integrity is controlled by the combined action of the VEGFR2 and MAP kinase pathways. Our results also suggest the possible side effect of the combination of two anticancer drugs on the circulatory system.     

Variations of matrix electrondensity in brain mitochondria.

The configuration of brain mitochondria was compared in situ, after aldehyde perfusion and/or osmium immersion fixation and in isolated fractions of different functional performance. After combined aldehyde perfusion osmium immersion fixation in situ, mitochondria were condensed having a dark matrix. Fractions capable of controlled respiration also consisted of condensed mitochondria. On the contrary, expanded mitochondria with light matrix were brought about by immersion fixation. Fractions consisting predominantly of light mitochondria displayed no controlled respiration. Light matrix and expanded form are therefore regarded as a functionally impaired state of brain mitochondria. The condensed form is thought to be a landmark of good fixation.     

Protein bodies in ray cells of Populus x canadensis Moench ‘robusta’

Light- and electron-microscopical investigations revealed distinct intravacuolar protein aggregates of 0.3–0.8 m in diameter in ray cells of poplar during the dormant season. In semi-thin sections, these bodies showed positive protein staining and enzymatic digestibility with pepsin, indicating their proteinaceous nature. Morphometric measurements showed such protein bodies in 7–13% of the area of the ray-cell lumen. This amount corresponded with the protein content of the wood determined biochemically, e.g. 2.0–5.0 g·mg^-1 dry weight. Polyacrylamide gel electrophoresis of the total protein fraction extracted from wood showed prominent polypeptide species with an apparent molecular weight of 30–32 kilodaltons. The results indicate considerable protein storage in ray cells, especially in the form of protein-storage vacuoles.Abbreviation DW dry weight     

Retention of lead within the digestive vacuole inTetrahymena

Summary The ciliateTetrahymena pyriformis was exposed to lead acetate. Cell proliferation in the presence of 0.1% lead salt (with or without EDTA) equaled, after a variable lag period, that of the control cells. The lead (550 ppm) forms a fluffy precipitate with the organic growth medium; this was in part prevented by addition of EDTA. The cells primarily ingested the fluffy precipitate whereby they became exposed to large amounts of lead. Within the digestive vacuole, the fluffy precipitate became converted into refractile structures (3 m in diameter) which were egested and accumulated at the bottom of the culture flask. The lead content of these defecation balls was higher than that of the fluffy precipitate. In addition to the lead-containing vacuoles, the cells contained small, refractile granules. The apparent, high tolerance ofTetrahymena towards lead is believed to be due in part to the low ionic concentration of lead under the present conditions and in part to a detoxication mechanism consisting of retention of lead within the digestive vacuoles and perhaps of accumulation of lead within the small, refractile granules.     

Plasmatubules: fact or artefact?

Plasmatubules are tubular evaginations of the plasmalemma associated with sites where high solute flux occurs between apoplast and symplast. Plasmatubules of the scutellar epithelial cells of germinating barley (Hordeum vulgare L.) have been examined following a variety of fixation methods. Of the aqueous fixations, primary aldehyde fixation with osmium post-fixation and osmium as the primary fixative gave comparable images, whilst potassium permanganate resulted in some distortion of the tissue in general including dilation of the tubular evaginations of the plasmalemma. Freeze-fixation and substitution with acetone and acetone-osmium gave images of the plasmalemma comparable to those obtained by the aqueous aldehyde and osmium methods. The similarity of structure with aldehyde or osmium and freezing as the primary fixation is taken to indicate that plasmatubules are real and not artefacts resulting from the fixation procedure.     

Effect of carbamylcholine on Harderian gland morphology in rats

Summary To determine the effect of cholinergic secretagogue on the Harderian gland of rats, several light- and electron-microscopic parameters were morphometrically assessed at different time intervals after carbamylcholine injection. In controls, two types of glandular cells (type A cells having 40–55 large vacuoles per cell profile and type B cells containing 30–38 smaller vacuoles per cell profile) and myoepithelial cells were recognized. At 5 min after injection of carbamylcholine, when rats secreted bloody tears, many alveoli showing narrower lumina and exocytotic figures in both types of cells were observed. Some vacuoles, which were covered by thin cytoplasmic sheets, protruded into the alveolar lumina. However, there was no evidence of apocrine or holocrine secretion. At 30 min and 120 min after injection, most of the alveolar lumina were dilated, and a pronounced decrease in the number of vacuoles in the glandular cells was observed. At 300 min after injection, the secretory vacuoles in both cell types reaccumulated. Transitional forms between the two cell types were not observed. The two types of Harderian gland cells can therefore be considered independent populations rather than different secretory stages of the same cell. It appears that the secretory process of the Harderian gland of rat is affected by cholinergic stimulation of the two types of glandular cells and of myoepithelial cells.     

Electron microscopy of the paracervical (Frankenhäuser) ganglion of the adult rat

Summary The ultrastructure of the paracervical (Frankenhäuser) ganglion in the rat was studied after immersion or perfusion fixation with glutaraldehyde followed by post-osmification. This ganglion is located at the uterovaginal junction in the vicinity of arteria uterina and contains three neuronal cell types. (1) Principal neurons have a fine structure mainly similar to the ganglion cells of other autonomic ganglia. (2) Small granule-containing cells occur in clusters often close to fenestrated capillaries. They are divided into two subgroups according to the size of their cytoplasmic granules; those containing only small granulated vesicles of 800 to 1400 Å in diameter and those having also large granulated vesicles of 2000 to 3000 Å in diameter. (3) Vacuolated nerve cells are large cells that resemble the principal neurons in their cytoplasmic components, except that they contain one to ten vacuoles with corpuscles of different size and shape. The possible physiological significance of the small, granule-containing cells in the uterine function is discussed.     

Endocytosis,digestive vacuolar movement and exocytosis on refeeding starvedTetrahymena pyriformis GL-9

Summary Evidence is presented in support of the hypothesis that the contents of digestive vacuoles in refed starvedTetrahymena pyriformis GL-9 are egested from the cell in approximately the sequence of their order of formation. The investigations involved measurements of the rates of disappearance of digestive vacuoles from the cells and the subsequent appearance of egested globules in the surrounding medium using both cultures and individual cells. The cells were first fed peptone and latex particles for a period and then this type of vacuole formation was suppressed by the addition of excess carmine particles (or the process was repeated with the particles in reverse order). Thus two types of morphologically distinct digestive vacuoles could be produced and observed microscopically. These observations suggest that the temporal nature of the movement of the digestive vacuoles through the cell result in the temporal nature of egestion and that no selective mechanism occurs at egestion. Thus digestive vacuoles are thought to pass through the cell from cytopharynx to cytoproct in approximately the order formed and at approximately constant rate. Under conditions of excess nutrients, where the cells become filled with digestive vacuoles, they seem to be able to maintain an approximately uniform number of digestive vacuoles within themselves by maintaining approximately constant and equal rates of vacuole formation and egestion. The maximum rates of latex or carmine vacuole formation or egestion found in single cells were approximately 0.3–0.4 vacuoles per cell per minute. The results are discussed.     

斑马鱼管腔器官空腔形态发生及分子机制

后生动物复杂的体内结构和器官结构多以网络状的管道系统出现。中空的管腔作为这个系统的重要结构单元承担了运输物质、区分器官不同部位功能、分隔机体和外环境等诸多重要的生理功能。管腔的发育障碍将致使相关器官形态发生畸形、功能紊乱。管腔型器官形态发生易被直接观察以及各种相关突变鱼和荧光转基因鱼的出现, 使得斑马鱼(Danio rerio)成为管道器官研究的优秀模式动物。斑马鱼血管、神经管、小肠、胰腺外分泌腺、前肾管等几种重要的器官的形态发生都伴随着典型的腔道发育过程, 是研究管腔形成的重要器官模型。管腔形成由胞外信号诱导、细胞极性化、胞内物质定向运输、腔内液体形成和胞内细胞骨架重构等相关管腔细胞内外发生的结构功能变化过程所构成, 而这些结构与功能的变化过程是通过精确而复杂的分子调控网络来实现, 最终形成管道器官。文章对斑马鱼4种典型管腔型器官的空腔形态发生过程进行了综述, 并总结了此过程中的分子机制, 为今后的相关研究提供了参考。     

Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo

The formation of intersegmental blood vessels (ISVs) in the zebrafish embryo serves as a paradigm to study angiogenesis in vivo. ISV formation is thought to occur in discrete steps. First, endothelial cells of the dorsal aorta migrate out and align along the dorsoventral axis. The dorsal-most cell, also called tip cell, then joins with its anterior and posterior neighbours, thus establishing a simple vascular network. The vascular lumen is then established via formation of vacuoles, which eventually fuse with those of adjacent endothelial cells to generate a seamless tube with an intracellular lumen. To investigate the cellular architecture and the development of ISVs in detail, we have analysed the arrangement of endothelial cell junctions and have performed single cell live imaging. In contrast to previous reports, we find that endothelial cells are not arranged in a linear head-to-tail configuration but overlap extensively and form a multicellular tube, which contains an extracellular lumen. Our studies demonstrate that a number of cellular behaviours, such as cell divisions, cell rearrangements and dynamic alterations in cell-cell contacts, have to be considered when studying the morphological and molecular processes involved in ISV and endothelial lumen formation in vivo.     

Electron microscopic observations of the influence of different fixatives on the appearance of cellular ultrastructure

Summary The cells of the proximal convoluted tubules of the rat kidney, mouse hepatic parenchymal cells, and the juxtaglomerular cells (J. G. cells) of the afferent arteriole in rabbit kidney served as test tissues for a study of the modifications of structure produced by 14 different fixation procedures using osmium tetroxide, a chrome-osmium solution, glutaraldehyde, and formaldehyde as fixatives. The fixative solutions containing various buffers were used either alone (osmium tetroxide-containing compounds) or in various combinations (aldehyde fixation with postfixation in osmium tetroxide). The most conspicuous differences in appearance following different fixation procedures were noted in the cytosomes of renal proximal tubular cells and the granules in the J. G. cells. However, virtually all cytoplasmic organelles showed some modification of structure with different fixatives. The evidence indicated that the chemical composition of the buffer used with OsO₄ was an important factor which particularly affected the density of the cytosomal matrix in renal proximal tubular and J. G. cells. The density of the matrix of the cytosomes was, however, also influenced by the fixative itself as indicated by studies of aldehyde-fixed tissues. With these fixatives the ultrastructural appearance was not modified by the buffer. Clumping of nuclear chromatin along the nuclear membrane and around the nucleolus occurred following fixation in aldehyde and Dalton's chrome-osmium solution, whereas the chromatin was evenly distributed in the nucleus when buffered osmium tetroxide was utilized for fixation. A specific reaction of dichromate with the granule of the J. G. cells seemed to take place rendering these granules electron opaque, thereby facilitating their identification in the light and electron microscope. The findings were discussed with particular attention to the interaction of fixatives and buffers with various tissue components.Supported by grants from the Board of Swedish Life Insurance Companies, the Stiftelsen Therese och Johan Anderssons Minne, and grant No. AM-7919 from the National Institutes of Health, Bethesda, Maryland (USA).     

Role of luminal buffers in renal tubular acidification

Summary The acidification kinetics of artificial solutions containing buffers of different permeancy were studied in rat proximal tubules by means of stationary microperfusion techniques. Luminal pH changes were measured by antimony microelectrodes and used to calculate net rates of acidification and the approach to steady-state pH levels. For most buffer species, tracer efflux out of the lumen was compared with changes in buffer concentration as derived from calculations based on the Henderson Hasselbalch equation. Steady-state luminal pH was similar for most buffer systems studied. However, secretory hydrogen ion fluxes into the lumen were significantly higher for permeant than for less permeant buffers. The most likely explanation is that permeant buffers behave as open systems maintaining constant low diffusible acid levels in the lumen, whereas impermeant buffers behave as closed systems in which nonionized acid levels are maintained at higher levels. A behavior consistent with this thesis was directly demonstrated for glycodiazine and, to a lesser degree, for DMO. In contrast, phosphate and creatinine behave like buffers in a closed cystem. Characteristics of proximal tubular acidification, of buffer reabsorption, and the effect thereupon of carbonic anhydrase inhibitors are satisfactorily explained by an essential role of (1) hydrogen ion secretion, (2) pK differences, and (3) different permeance of the non-ionized buffer species. However, specific transport mechanisms may, in addition, also contribute to differences in transepithelial buffer movement.     

Synthesis of antiparallel 4α-helix bundle TASP by chemoselective ligation

Summary The use of chemoselective ligation methods and orthogonal protection techniques allows access to Template-Assembled Synthetic Protein (TASP) molecules exhibiting a large variety of packing topologies. This is demonstrated for the synthesis of an antiparallel 4-helical bundle TASP by condensing amphiphilic peptide blocks, containing aldehyde functions at the C- or N-terminus, to a selectively addressable topological template via oxime bond formation. The resulting antiparallel 4-helix TASP is obtained in high yield and shows a template-induced helical conformation.     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

The alveolar-lining layer in the lung of the axolotl,Ambystoma mexicanum

Summary Lungs of neotenic larvae of Ambystoma mexicanum were prepared for maintaining the air-tissue boundary during aldehyde fixation. Four methods of postfixation were applied: 1) osmium tetroxide followed by en-bloc staining with uranyl acetate and phosphotungstic acid, 2) ruthenium redosmium tetroxide, 3) osmium tetroxide-ferrocyanide, and 4) tannic acidosmium tetroxide.Three types of cells line the inner surface of the axolotl lung: 1) pneumocytes, covering the capillaries with flat cellular extensions and containing two types of granules: the osmiophilic lamellar bodies, precursors of extracellular membranous material, and apical granules of unknown significance; 2) ciliated cells, also containing osmiophilic lamellar bodies; and 3) goblet cells filled with secretory granules as well as osmiophilic bodies.The extracellular material forms membranous whorls as well as tubular myelin figures, consisting of membranous backbones combined with an intensely stained substance. This material strikingly resembles the surfactant of amphibian lungs.