Showing papers in "Journal of Cell Biology in 1961"

Improvements in epoxy resin embedding methods.

Abstract: Epoxy embedding methods of Glauert and Kushida have been modified so as to yield rapid, reproducible, and convenient embedding methods for electron microscopy. The sections are robust and tissue damage is less than with methacrylate embedding.

Satellite cell of skeletal muscle fibers.

Abstract: In the course of an electron microscopic study of the peripheral region of the skeletal muscle fiber of the frog, the presence of certain cells, intimately associated with the muscle fiber, have been observed which we have chosen to call satellite cells. Since these cells have not been reported previously and indeed might be of interest to students of muscle histology and furthermore, as we shall suggest, might be pertinent to the vexing problem of skeletal muscle regeneration, a brief communication describing this finding is warranted prior to a more detailed study. The observations reported here have been made on bundles of fibers dissected from the tibialis anticus muscle of the frog. The material has been fixed by the conventional method with osmium tetroxide, and the embedding has been carried out with methacrylate and with epoxy (epon) resin. In sections that were \"stained,\" the lead hydroxide solution of Watson (1) was used. As seen in the attached electron micrograph of the satellite cell, the striking paucity of cytoplasm relative to its nucleus results in the cell assuming the shape of the nucleus. In fact, it is virtually impossible to discern the cellular nature of this entity in the light microscope, as it appears to be indistinguishable from a peripheral muscle nucleus proper. In electron micrographs the cell is seen \"wedged\" between the plasma membrane of the muscle fiber and the basement membrane, which invests the fiber throughout its length in close association with the plasma membrane. The intimacy of this satellite cell with respect to the multinucleate muscle cell is further revealed in the fact that, in general, the surface of the muscle fiber is not distorted outward but instead the satellite cell protrudes inward pushing the myofibrils of the muscle cell aside. On the inner surface, the plasma membrane of the satellite cell is in appositon with the plasma membrane of the muscle cell. Unfortunately, because of the limited observations and the difficulty in acquiring sufficient data readily with electron micrographic techniques, it is not possible at present to estimate the frequency of occurrence of these cells in a typical muscle fiber in our preparation of tibialis anticus muscle. The only generalization warranted at this time is that the peripheral muscle nuclei proper occur much more frequently than the satellite cells. It is interesting that upon alerting other investigators to these findings, similar cells have been found in electron micrographs of two other muscles of the frog, namely sartorius (2) and ileofibularis (3), and of the sartorius and tongue muscle of the white rat (4). (Though the direct evidence is restricted to these two vertebrates, it seems reasonable to hazard a guess that skeletal muscle fibers of vertebrates in general contain satellite cells.) It is tempting to speculate about the origin and the role of the satellite cells. Before stating the several possible hypotheses that have figured in our interpretations, it is pertinent to recall a most striking characteristic of regenerating muscle fibers in the least ambiguous case where the sarcolemma-tube remains intact, the myoplasm having undergone hyaline formation and retraction as a result of trauma. Within 48 hours a marked presence of \"free cells\" is noted in the empty tube, the cells appearing both as \"round\" and \" f u s i f o r m \" types (5). Moreover, in tissue culture studies of mature skeletal muscle explants, fi'ee cells are also seen emanating from the explant. The central question must be asked: what is the origin of these cells? Most cytologists lean toward the interpretation that surviving nuclei in the damaged multinucleate muscle cell give rise to single cells by \"gathering up\" cytoplasm from the sarcoplasm of the muscle cell--an unusual mechanism, however, for vertebrate systems. If this point of view is taken, the first and immediate hypothesis suggests itself, namely, that in the resting state some cells are being produced at a slow rate by the above mechanism and reside just outside the plasma membrane of the muscle cell, and that upon being stimulated by trauma, e.g. ischemia, mechanical compression, toxic agents, etc., the rate of production of such cells is increased. The second hypothesis, more in keeping with conventional notions of cytology, is that the satellite cells are remnants from the embryonic development of the multinucleate muscle cell which results from the process of fusion of individual myoblasts. Thus the satellite cells are

STUDIES ON INFLAMMATION: I. The Effect of Histamine and Serotonin on Vascular Permeability: An Electron Microscopic Study

Abstract: The mechanism, whereby histamine and serotonin increase the permeability of blood vessels, was studied in the rat by means of the electron microscope. The drugs were injected subcutaneously into the scrotum, whence they diffused into the underlying (striated) cremaster muscle. An intravenous injection of colloidal HgS was also given, in order to facilitate the identification of leaks by means of visible tracer particles. After intervals varying from 1 minute to 57 days the animals were killed; the cremaster was fixed, embedded in methacrylate, and examined with the electron microscope. One to 12 minutes after the injection, the blood vessels of the smallest caliber (3 to 5 micra as measured on electron micrographs) appeared intact. Numerous endothelial openings were present in blood vessels with a diameter of 7 to 8 micra or more. These gaps were 0.1 to 0.8 micra in width; portions of intercellular junctions were often present in one or both of the margins. The underlying basement membrane was morphologically intact. An accumulation of tracer particles and chylomicra against the basement membrane indicated that the latter behaved as a filter, allowing fluid to escape but retaining and concentrating suspended particulate matter of the size used. Uptake of tracer particles by endothelial vesicles was minimal. Phagocytosis by endothelial cells became more prominent at 3 hours, but as a secondary occurrence; the pericytes were actively phagocytic at all stages. At the 3-hour stage no leaks were found. The changes induced by histamine and serotonin were indistinguishable, except that the latter was more potent on a mole-to-mole basis. In control animals only small accumulations of tracer particles were found in the wall of a number of blood vessels. With regard to the pathogenesis of the endothelial leaks, the electron microscopic findings suggested that the endothelial cells become partially disconnected along the intercellular junctions. Supporting evidence was provided at the level of the light microscope, by demonstrating-in the same preparation-the leaks with appropriate tracer particles(1), and the intercellular junctions by the silver nitrate method. The lipid nature of the chylomicron deposits observed in electron micrographs was also confirmed at the level of the light microscope, using cremasters fixed in formalin and stained in toto with sudan red.

A modified procedure for lead staining of thin sections.

Abstract: Meta l impregnat ion , or "s ta in ing ," of tissue sections for electron microscopy has become the accepted practice in recent years. Salts of metals of h igh atomic weight such as u ran ium, chromium, thor ium, lead, or tungsten, among many tested, have been found suitable (6, 1). "Lead hydroxide ," as prepar ted by Watson (6), is now widely used, hu t this solution is extremely unstable in air and becomes covered by a meta l l ic-appear ing film which imparts to the " s ta ined" section a well known deposit of e lectron-opaque particles and crystals, reducing considerably the percentage of clean areas suitable for micrography (Fig. l) . Several procedures and devices have been described to lessen these contaminat ions. Peachey (5), using lead hydroxide, or subacetate, found tha t if the solution is kept in a syringe with the protect ing cap filled with sodium hydroxide to absorb the CO2 of the atmosphere, con tamina t ion is reduced. More recently, a fairly unwieldy appara tus has been suggested for the same purpose (4). Tests performed in our laboratory in which " s t a in ing" was a t tempted in a chamber under cont inuous ni t rogen flow and in the presence of a ba r ium chloride t rap were not completely successful, which is to say tha t con tamina t ion cont inued to be a problem. A n u m b e r of o ther var iat ions have been recommended. Lever (2), e.g., described a method for prepar ing a " s t a in ing" solution by adding potassium hydroxide to a lead hydroxide solution. To dissolve some of the occasionally formed crystals of contamina t ion , the sections are rinsed afterwards for a few seconds in a weak potassium hydroxide solution; this step is very critical because the "s ta in ing" is also weakened by the alkali and uni form staining is difficult to achieve. These various shortcomings of current ly avai lable methods have st imulated us to search for a lead salt tha t would not be affected by the components of the atmosphere. Among several heavy metal salts tested, the above-ment ioned " lead hydroxide" of Watson seemed to be the most effective in "s ta in ing ." Exper iments with the commercial ly available product showed that this salt would give no impregna t ion when dissolved in water, or in Institute. Dr. Millonig's present address is Biological

Simple methods for "staining with lead" at high pH in electron microscopy.

Abstract: The lead hydroxide stain of Watson (1958) used for increasing contrast in thin sections for electron microscopy has found acceptance in many laboratories. However, this stain has an unfortunate tendency to form precipitates (probably of lead carbonate (5)) on exposure to the air, thus contaminating the sections and irritating the observer. This drawback has led to the development of several modifications (2, 3) of the original method of staining and the use of ingenious devices (4, 5) for preventing exposure to air and consequent precipitate formation. We offer the following alternative methods which, we believe, are simpler to perform than those hitherto described. They have the additional advantages mentioned below. The methods are based on the observation that highly alkaline solutions of lead salts (pH > 11.5) yield relatively stable solutions which stain rapidly and intensely, thus obviating the hazard of precipitation to a marked degree. The methods have these additional advantages: the staining solutions are easily and rapidly prepared, are simply stored, and are stable for long periods of time. Furthermore, they can be efficiently used, many grids being treated simultaneously, without excessive precautions being taken against lead carbonate precipitation. Finally, \"difficult\" material, embedded in media which characteristically yield rather low contrast, such as epoxide resins, can be rapidly and easily stained. \"C lean\" preparations, of high contrast, are routinely obtained. As will be discussed later, it is thought that in these highly alkaline staining solutions lead is present as an hydroxide complex anion (plumbite ion) and that this anion is responsible for the staining. The methods of preparation are based on this hypothesis. Two methods for preparing the staining solutions have been found useful:

Mitochondrial localization of oxidative enzymes: staining results with two tetrazolium salts.

Abstract: A comparison is made of the staining results obtained with Nitro-BT and MTT-Co++ as acceptors when DPNH is the substrate in frozen sections of cold formol-calcium-fixed rat kidney (normal and following ligation of the blood vessels) and human liver containing lipofuscin granules. The kidney results are evaluated in terms of mitochondrial morphology seen after classical mitochondrial stains and in electron micrographs. It is concluded that, except for formazan deposition on lipid-aqueous interfaces, Nitro-BT staining indicates the intracellular localization of oxidative enzymes, at least at the level of light microscopy. In contrast, the use of MTT-Co++ is not reliable for such intracellular localizations. The deposition of the formazan of MTT-Co++ is determined in large part by physicochemical factors other than enzyme localization. Despite marked abnormalities of the mitochondria in cells of the ligated kidney, MTT-Co++ formazan is generally deposited in the same dotlike fashion as in cells of normal kidney.

STUDIES ON INFLAMMATION : II. The Site of Action of Histamine and Serotonin along the Vascular Tree: A Topographic Study

Abstract: While it is an established fact that histamine and serotonin increase the permeability of blood vessels, the exact portion of the vascular tree which is so affected has not been conclusively demonstrated. The present study was undertaken to clarify this point. Our experiments were based on a method to which we refer as "vascular labeling," and which permits one to identify leaking vessels by means of visible accumulations of foreign particles within their walls. The mechanism of the labeling, elucidated by previous electron microscopic studies, is the following. Histamine and serotonin cause the endothelial cells of certain vessels to separate, and thus to create discrete intercellular gaps. Plasma escapes through these gaps, and filters through the basement membrane. If the plasma has been previously loaded (by intravenous injection) with colloidal particles of a black material such as carbon or mercuric sulfide, these particles—too large to pass through the basement membrane—will be retained and accumulate in visible amounts within the wall of the leaking vessel. This method is used to maximal advantage if the tissue is cleared and examined by transillumination in toto, so that leaking vessels can be accurately identified in their relationship to the vascular tree. As a test tissue we used the rat cremaster, a laminar striated muscle which can be easily excised with its vascular supply virtually intact. The rats were prepared with an intravenous injection of carbon or HgS, and a subcutaneous injection into the scrotum of histamine, serotonin, or NaCl (as a control). The injected drug diffused into the underlying cremaster and the vessels became labeled. One hour later, when the carbon had been cleared from the blood stream, the animal was killed. The cremaster was excised, stretched, fixed in formalin, cleared in glycerin, and examined by transillumination under a light microscope. The lesions induced by histamine and serotonin were identical. The leaking vessels, as indicated by the carbon deposits, always belonged to the venous side of the circulation. The heaviest deposits were found in venules 20 to 30 micra in diameter. The deposits decreased towards larger venules up to a maximum diameter of 75 to 80 micra, and towards the finer vessels until the caliber reached approximately 7 micra. Essentially spared by the deposits were the finest vessels, 4 to 7 micra in diameter, and constituting an extensive network oriented along the muscular fibers. By killing animals at varying intervals after the injections, it was found that the carbon particles were slowly removed from the vascular walls by the action of phagocytic cells. After 10 months there was still enough carbon locally to be recognized by the naked eye.

A simple method for removing the resin from epoxy-embedded tissue.

Abstract: Epoxy resins were first used as embedd ing media for electron microscopy by MaalCe and BirchAndersen (1) and Glauer t and Glauer t (2) and have since been employed extensively in Grea t Bri tain by Huxley (3) and Rober tson (4). Recen t improvements in processing and embedd ing techniques by Luft (5) and Finck (6) have led to their acceptance in many laboratories throughout the Uni t ed States. In correlated studies using electron and l ight microscopy it is usually necessary to remove the embedd ing mater ia l from the thick sections dest ined for convent ional l ight microscopy before they yield opt imal results in cellular detail and s taining qualities. Both xylol and acetone are excellent and rapid solvents for methacrylate , and removal of this plastic and subsequent staining for l ight microscopy are simple procedures (Bencosme et al., 7). Epoxy resins, however, are not soluble in s tandard organic solvents. Fisch and Hofmann (8) have studied the controlled chemical degrada t ion into soluble components of epoxy resins cured with phthal ic anhydride. Applying some of their findings we have developed a suitable solvent for use in tissue-resin systems.

Ultrastructural study of remyelination in an experimental lesion in adult cat spinal cord

Abstract: This report presents ultrastructural observations on the cytological events that attend myelin formation occurring in the wake of demyelination in adult cat spinal cord Lesions were induced in subpial cord by cerebrospinal fluid (csf) exchange (1, 2) Tissue from eleven cats at nine intervals from 19 to 460 days was fixed in situ by replacing csf with buffered OsO4 and embedded in Araldite After demyelination, axons are embraced by sheet-like glial processes An occasional myelin sheath is first seen at 19 days; by 64 days, all axons are at least thinly myelinated The cytoplasm of the myelin-forming cells, unlike that of either oligodendrocyte or fibrous astrocyte in normal cord, is dense with closely packed organelles and fine fibrils Many of the myelinogenic cells become scarring astrocytes and at 460 days the lesion teems with their fibril-filled processes Oligodendrocytes appear in the lesion after remyelination is under way Phagocytes disappear gradually A myelin sheath is formed by spiral wrapping of a sheet-like glial process around an axon Where the first turn of the spiral is completed, a mesaxon is formed As cytoplasm is lost from the process, the plasma membrane comes together along its outer and cytoplasmic surfaces to form compact myelin Only a small amount of cytoplasm is retained; it is confined to the paramesaxonal region and, on the sheath exterior, to a longitudinal ridge which appears in profile as a small loop This outer loop has the same rotational orientation as the inner mesaxon These vestiges of spiral membrane wrapping are also found in normal adult and new-born cat cord Nodes are present in all stages of remyelination and in normal adult cat and kitten cord These observations suggest that myelin is reformed in the lesion in the same way it is first formed during normal development The mechanism of myelin formation is basically similar to that proposed for peripheral nerve and amphibian and mammalian optic nerve; it does not agree with present views on the mechanism of myelinogenesis in mammalian brain and cord This is the first demonstration of remyelination in adult mammalian central nervous tissue

Preferential staining of nucleic acid-containing structures for electron microscopy

Abstract: Oriented fibres of extracted nucleohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tetroxide solution at pH 6, containing 10(-2)M Ca(++), and embedding in Araldite enabled sections of the fibres to be cut in which the orientation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqueous solution. Strongest staining of whole tissue was obtained with very short fixation times-5 minutes or so at 0 degrees C. Particularly intense staining was obtained when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissues fixed for longer times ((1/2) hour to 2 hours, at 0 degrees C or 20 degrees C), in briefly fixed material the DNA-containing regions appeared relatively unstained by lead hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.

The relationship between the fine structure and direction of beat in gill cilia of a lamellibranch mollusc

Abstract: This paper describes the fine structure and its relationship to the direction of beat in four types of cilia on the gill of the fresh-water mussel Anodonta cataracta. The cilia contain nine outer, nine secondary, and two central fibers, such as have been described previously in other material. Each outer fiber is a doublet with one subfiber bearing arms. One particular pair of outer fibers (numbers 5 and 6) are joined together by a bridge. The two central fibers are enclosed by a central sheath; also present in this region is a single, small mid-fiber. The different groups of fibers are connected together by radial links that extend from the outer to the secondary fibers, and from the secondary fibers to the central sheath. The basal body consists of a cylinder of nine triplet fibers. Projecting from it on one side is a dense conical structure called the basal foot. The cylinder of outer fibers continues from the basal body into the cilium, passing through a complex transitional region in which five distinct changes of structure occur at different levels. There are two sets of fibers associated with the basal bodies: a pair of striated rootlets that extends from each basal body down into the cell, and a system of fine tubular fibers that runs parallel to the cell surface. The relationship between fine structure and direction of beat is the same in all four types of cilia examined. The plane of beat is perpendicular to the plane of the central fibers, with the effective stroke toward the bridge between outer fibers 5 and 6, and toward the foot on the basal body.

A new freezing-ultramicrotome.

Abstract: The difficulties in sectioning frozen biological objects for electron microscopic investigations are overcome by Steere's freezing-etching method. In order to test this method and to open up a wide field of application, the new freezing-ultramicrotome has been designed. The apparatus consists of the combination of an ultramicrotome with freezing-drying and shadow-casting installations in the same vacuum container. The preliminary results show, on the one hand, the practicability of all preparational steps and, on the other, that it is possible to resolve internal structures of cell organelles and even macromolecular patterns.

Wound healing and collagen formation. I. Sequential changes in components of guinea pig skin wounds observed in the electron microscope.

Abstract: The regular sequence encountered in healing guinea pig skin wounds has been examined by methods of light and electron microscopy. Observations on cell populations, their fine structure, and fibril formation in the connective tissue have been made. Linear incisions in the skin of normal female guinea pigs weighing 300 to 350 grams were allowed to heal. The wounds were then excised, fixed with buffered 2 per cent osmium tetroxide, and postfixed in neutral buffered formalin, at 16 and 24 hours and at 3, 5, 9, and 14 days after wounding. They were then embedded in epoxy resin. In the inflammatory phase the exudate observed in the early wounds consists largely of polymorphonuclear neutrophilic leukocytes, macrophages, fibrin, and free extracellular organelles from the disrupted inflammatory cells. These organelles later appear in vacuoles in the cytoplasm of the macrophages. Fibroblasts first appear at 24 hours, and show extensive development and dilatation of the endoplasmic reticulum, which sometimes contains moderately dense flocculent material. In addition, these fibroblasts have enlarged mitochondria and condensations of filamentous material within the cytoplasm near the cell surface. Occasional myelin figures and moderately dense, 0.5 to 1.0 micron bodies are found within the cytoplasm of the early fibroblasts. Collagen fibrils are first seen at 3 days extracellularly near the cell surfaces. They appear at the later times in two populations of sizes. With increasing wound age the fibroblasts retain their morphology and the wounds decrease in cellularity concomitantly with the formation of increasing amounts of collagen. Several proposed mechanisms of collagen fibril formation are discussed in relation to the observed phenomena. The problem of correlating fibril diameter with the appearance of the periodic structure of collagen in relation to the minimal size fibril which would be anticipated to display this appearance is discussed.

Pore canals and related structures in insect cuticle.

Abstract: The fine structure and the distribution of an esterase have been studied in the cuticle of Galleria larvae, Tenebrio larvae and pupae, and in the wax-secreting cuticle of the honey bee, and compared with those in the cuticle of the caterpillar of Calpodes. In Galleria and Tenebrio the pore canals are spaces passing through the lamellate endocuticle from the epithelium to the epicuticle. They contain a filament from the cells which may be concerned in their formation. The shape of the pore canal is probably determined by the orientation of the fibres making up the lamellae in the endocuticle and is not a regular helix. The pore canals also contain numerous filaments of another sort which pass on through the epicuticle and are believed to be the origin of the surface wax. They are particularly abundant in the pore canals of the honey bee wax-secreting cuticle and extend into the cell in long pockets surrounded by an envelope of the plasma membrane. The esterase is probably concerned with the final stage of wax synthesis, for its distribution is similar to that of the lipid filaments.

The development of vaccinia virus in Earle's L strain cells as examined by electron microscopy.

Abstract: A favorable system which is amenable to frequent and reproducible sampling, consisting of suspension cultures of strain L cells and vaccinia virus, was employed to study the animal virus-mammalian host cell relationship. The three principal aspects investigated concerned the adsorption and penetration of vaccinia into the host, the relationship between the sequence of virus development and the production of infectious particles, and the changes in the fine structure of the host cells. Experiments in which a very high multiplicity of infection was used revealed that vaccinia is phagocytized by L cells in less than 1 hour after being added to the culture, without any apparent loss of its outer limiting membranes. Regions of dense fibrous material, thought to be foci of presumptive virus multiplication, appear in the cytoplasm 2 hours after infection. A correlation between electron microscope studies and formation of infectious particles shows that although immature forms of the virus appear 4 hours after infection, infectious particles are produced 6 hours after infection of the culture, at the time when mature forms of vaccinia appear for the first time in thinly sectioned cells. Spread of the infection is gradual until eventually, after 24 hours, virus is being elaborated throughout the cytoplasm. Addition of vaccinia to monolayer cultures induced fusion of L cells and rapid formation of multinucleate giant forms. In both suspension and stationary cultures infected cells elaborate a variety of membranous structures not present in normal L cells. These take the form of tube-like lamellar and vesicular formations, or appear as complex reticular networks or as multi-laminar membranes within degenerating mitochondria.

The properties of specific stains for electron microscopy prepared by the conjugation of antibody molecules with ferritin

Abstract: In order to take full advantage of recent developments in the electron microscopic examination of cellular ultrastructure and composition, it is necessary to develop specific electron stains capable of identifying and localizing a wide variety of macromolecular components of cells. To this end, antibody conjugates have been prepared by chemically coupling the highly electron-scattering ferritin molecule to antibody. Antigen-antibody precipitations with these ferritin-antibody conjugates have demonstrated that under the appropriate conditions they retain the specific binding properties of the antibody from which they are prepared. An electron microscopic study has been made of aggregates of tobacco mosaic virus and its ferritin-conjugated antibody. The aggregates were prepared in solution and then sprayed onto specimen screens. The electron micrographs reveal that the conjugate specifically attached to, and delineated, the virus rods. The chemistry, structure, and resolving power of the ferritin-antibody conjugates, the specificity of their reactions with homologous antigen, and the nature of the problems to be faced in application of these conjugates to the study of the internal antigens of cells are discussed.

The normal fine structure of opossum testicular interstitial cells

Abstract: The interstitial tissue of the opossum testis includes interstitial or Leydig cells, macrophages, and small cells which morphologically resemble mesenchymal cells. The latter are thought to give rise to mature interstitial cells. The most prominent feature of the interstitial cell cytoplasm is an exceedingly abundant agranular endoplasmic reticulum. This reticulum is generally in the form of a meshwork of interconnected tubules about 300 to 450 A in diameter, but occasionally it assumes the form of flattened, fenestrated cisternae resembling those of pancreatic acinar cells, except for the lack of ribonucleoprotein particles on the surface of the membranes. The interstitial cells vary considerably in their cytoplasmic density. The majority are quite light, but some appear extremely dense, and in addition usually have a more irregular cell surface, with numerous small pseudopodia. These differences may well reflect variations in physiological state. Cytoplasmic structures previously interpreted as "crystalloids" consist of long bundles of minute parallel tubules, each about 180 A in diameter, which seem to be local differentiations of the endoplasmic reticulum. The mitochondria are rod-shaped, and contain a moderately complex internal membrane structure, and also occasional large inclusions that are spherical and homogeneous. The prominent juxtanuclear Golgi complex contains closely packed flattened sacs and small vesicles. The results of the present study, coupled with biochemical evidence from other laboratories, make it seem highly probable that the agranular endoplasmic reticulum is involved in the synthesis of the steroid hormones produced by the interstitial cell. This finding therefore constitutes one of the first functions of the agranular reticulum for which there is good morphological and biochemical evidence.

The localization of acid phosphatase in rat liver cells as revealed by combined cytochemical staining and electron microscopy

Abstract: Discrete localization of stain in pericanalicular granules was found in 10 µ frozen sections of formol-phosphate-sucrose-fixed liver stained by the Gomori acid phosphatase technique and examined in the light microscope. The staining patterns, before and after treatment with Triton X-100 and lecithinase, were identical with those previously reported for formol-calcium-fixed material treated in the same way, and it can be assumed that the stained granules are identical with "lysosomes." Examination in the light microscope of the staining patterns and lead penetration in fixed blocks and slices of various dimensions showed nuclear staining and other artefacts to be present, produced by the different rates of penetration of the various components of the staining medium into the tissue. A uniform pericanalicular staining pattern could be obtained, however, with slices not more than 50 µ thick, into which the staining medium could penetrate rapidly from both faces. The staining pattern produced in 50 µ slices was the same both at pH 5.0 and pH 6.2, and was not altered by subsequent embedding of the stained material in butyl methacrylate. Electron microscopy showed the fine structure of fixed 50 µ frozen slices to be well preserved, but it deteriorated badly when they were incubated in the normal Gomori medium at pH 5.0 before postfixing in osmium tetroxide. After incubation in the Gomori medium at pH 6.2, the detailed morphology was substantially maintained. In both cases lead phosphate, the reaction product, was found in the pericanalicular regions of the cell, but only in the vacuolated dense bodies and never in the microbodies. Not every vacuolated dense body contained lead, and stained and unstained bodies were sometimes seen adjacent to each other. This heterogeneous distribution of stain within a morphologically homogeneous group of particles is consistent with de Duve's suggestion (9) that there is a heterogeneous distribution of enzymes within the lysosome population. It is concluded from these investigations that the vacuolated dense bodies seen in the electron microscope are the morphological counterparts of the "lysosomes" defined biochemically by de Duve.

Structure of the toad's urinary bladder as related to its physiology.

Abstract: The structure of the urinary bladder of the toad Bufo marinus was studied by light and electron microscopy. The epithelium covering the mucosal surface of the bladder is 3 to 10 microns thick and consists of squamous epithelial cells, goblet cells, and a third class of cells containing many mitochondria and possibly representing goblet cells in early stages of their secretory cycle. This epithelium is supported on a lamina propria 30 to several hundred microns thick and containing collagen fibrils, bundles of smooth muscle fibers, and blood vessels. The serosal surface of the bladder is covered by an incomplete mesothelium. The cytoplasm of the squamous epithelial cells, which greatly outnumber the other types of cells, is organized in a way characteristic of epithelial secretory cells. Mitochondria, smooth and rough surfaced endoplasmic reticulum, a Golgi apparatus, "multivesicular bodies," and isolated particles and vesicles are present. Secretion granules are found immediately under the plasma membranes of the free surfaces of the epithelial cells and are seen to fuse with these membranes and release their contents to contribute to a fibrous surface coating found only on the free mucosal surfaces of the cells. Beneath the plasma membranes on these surfaces is an additional, finely granular component. Lateral and basal plasma membranes are heavily plicated and appear ordinary in fine structure. The cells of the epithelium are tightly held together by a terminal bar apparatus and sealed together, with an intervening space of only 0.02 mµ near the bladder lumen, in such a way as to prevent water leakage between the cells. It is demonstrated in in vitro experiments that water traversing the bladder wall passes through the cytoplasm of the epithelial cells and that a vesicle transport mechanism is not involved. In vitro experiments also show that the basal (serosal) surfaces of the epithelial cells are freely permeable to water, while the free (mucosal) surfaces are normally relatively impermeable but become permeable when the serosal surface of the bladder is treated with neurohypophyseal hormones. The permeability barrier found at the mucosal surface may be represented, structurally, either by the filamentous layer lying external to the plasma membrane, by the intracellular, granular component found just under the plasma membrane, or by both of these components of the mucosal surface complex. The polarity of the epithelial sheet is emphasized and related to the physiological role of the urinary bladder in amphibian water balance mechanisms.

Localization of acid phosphatase activity in hepatic lysosomes by means of electron microscopy

Abstract: Samples of liver from untreated rats, from rats infused with unconjugated bilirubin, and from biopsies of human liver were fixed overnight in cold formol-calcium. Frozen sections were stained for acid phosphatase activity by the Gomori lead-glycerophosphate procedure. Small blocks of fixed tissue were also incubated in this medium. These were then treated briefly with osmium tetroxide, dehydrated, and embedded in methacrylate. Thin sections were studied by electron microscopy. The sites of reaction product of acid phosphatase activity as visualized in electron micrographs are consistent with those seen in frozen sections studied by light microscopy. They indicate that the pericanalicular bodies of parenchymatous cells, the large spherical bodies of Kupffer cells, the microbodies appearing after bilirubin infusion and lipofuscin granules belong to the class of cytoplasmic organelles called lysosomes by de Duve.

Studies on formalin fixation for electron microscopy and cytochemical staining purposes.

Abstract: A study has been made of the preservation of fine structure, phospholipids, and the activity of acid phosphatase and esterase in rat liver fixed in various solutions containing 4 per cent formaldehyde. Examination of methacrylate-embedded preparations shows that calcium-containing fixatives result in poor preservation of fine structure, whereas veronal-treated or phosphate-buffered formalin gives excellent results if the tonicity of the solutions is suitably adjusted by addition of sucrose. Formol-phosphate, to which Versene has been added, causes deterioration of cellular morphology. Phospholipids are retained almost quantitatively in tissue fixed in formol-calcium, and in phosphate-, collidine-, or triethanolamine-buffered formalin. About 50 per cent of the activity of acid phosphatase and esterase are preserved after 24 hours exposure to these fixatives at 0–2°C, and the distributions of the enzymes and of phospholipids, as judged by cytochemical staining results, are not altered by any of these formalin solutions. Consideration of the morphological and biochemical integrity of the fixed tissue suggests that 4 per cent formaldehyde, buffered at pH 7.2 with 0.067 M phosphate, and containing 7.5 per cent sucrose, is the most suitable of the fixatives for combined cytochemical staining and electron microscopical studies.

The fine structure of bone cells

Abstract: An electron microscopic study of Araldite-embedded, undecalcified human woven and chick lamellar bone is presented. The fine structure of the cells of bone in their normal milieu is described. Active osteoblasts possess abundant granular endoplasmic reticulum, numerous small vesicles, and a few secretion droplets. Their long cytoplasmic processes penetrate the osteoid. The transition of osteoblasts into osteoid osteocytes and then into osteocytes is traced and found to involve a progressive reduction of cytoplasmic organelles. Adjoining the osteocytes and their processes is a layer of amorphous material which is interposed between the cell surfaces and the bone walls of their respective cavities. Osteoclasts contain numerous non-membrane-associated ribosomes, abundant mitochondria, and little granular endoplasmic reticulum, thus differing markedly from other bone cells. The brush border is a complex of cytoplasmic processes adjacent to a resorption zone in bone. No unmineralized collagen is seen at resorption sites and it appears that collagen is removed before or at the time of mineral solution. All bone surfaces are covered by cells, some of which lack distinctive qualities and are designated endosteal lining cells. The structure of osteoid, bone, and early mineralization sites is illustrated and discussed.

The sarcoplasmic reticulum of a fast-acting fish muscle.

Abstract: In the early years of this century Vera t t i (15) and a few other uncommonly observant cytologists (11) demonst ra ted in the sarcoplasm of striated muscle a delicate plexus of strands sur rounding all of the myofibrils. The transverse elements of this network were located in a constant relat ion to the crossbanded pat tern of the myofibrils. The majori ty of cytologists of tha t period seem not to have accepted the reality of this network, possibly because of their mistrust of the capricious metallic impregnat ion methods required for its demonstration. Therefore, it received little fur ther study unti l some 50 years later when Bennet t and Porter (3) and Andersson (1) directed a t tent ion to a vacuolar or tubu la r interfibri l lar componen t of the sarcoplasm visible in electron micrographs of striated muscle. Extending these observations, Porter and Palade (9) later demonst ra ted tha t these membranous profles are par t of an elaborate reticular system of anastomosing tubules tha t surrounds all the myofibrils. The majori ty of the tubu la r elements of this sarcoplasmic reticulum run longitudinally, bu t there is, for each muscle, a characterist ic repeat ing pa t te rn of transverse channels occurr ing in register with par t icular bands in the cross-striations of the myofibrils. These transverse components , called the \" t r iads\" of the ret iculum, consist of pairs of parallel tubes separated by an intermediate row of small vesicles. The continuous na ture of the ret iculum, its in t imate relat ion to the myofibrils and to the cell surface have led to the suggestion tha t the membranes of this system may be involved in impulse conduct ion from the sarcolemma to the contractile elements in the interior of the muscle fiber (4, 7, 9). Compara t ive studies by Peachey and Porter (7) correlat ing cell size, speed of contraction, and degree of development of the reticulum have provided evidence tending to support this hypothesis. The investigation reported here extends these comparat ive observations on the sarcoplasmic re t iculum to a par t icular ly fastacting fish muscle. A n u m b e r of teleost fish including the sea robins (Prionotus), the toadfish (Opsanus), the croakers (Micropogon) and drums (Sciaenops) are capable of making clearly audible sounds by a variety of mechanisms. The deep resonant sounds of the toadfish are made by rapid contract ion of the intrinsic striated muscle in the taut wall of its gas-filled swim-bladder (14). In recent studies of the neuromuscular mechanism of sound production in this species, Skoglund (12) has shown tha t the muscle involved reaches its contract ion peak and returns to complete relaxat ion very much more rapidly than do most ver tebra te muscles. The present paper describes an elaborate development of the sarcoplasmic re t iculum and other fine structural features of this muscle which may be related to its unusual physiological properties.

The route of entry and localization of blood proteins in the oocytes of saturniid moths.

Abstract: The oocytes of saturniid moths take up proteins selectively from the blood. The distribution of blood proteins in the ovary during protein uptake was investigated by staining 2 µ sections of freeze-dried ovaries with fluorescein-labeled antibodies. The results indicate that blood proteins occur primarily in the intercellular spaces of the follicle cell layer, in association with a brush border at the surface of the oocyte, and within the oocyte in the yolk spheres. That proteins derived from the blood are associated with the yolk spheres was confirmed by isolating these bodies and showing that lysis, which can be induced by any of a number of mechanical means, causes them to release immunologically defined proteins known to be derived from the blood. That the level of blood proteins in the cytoplasm is low relatively to that in the yolk spheres was confirmed by the observation that the yellow pigments associated with several blood proteins, although conspicuous in the yolk spheres, are not visible in the translucent layer of centrifuged oocytes. From these and previous physiological observations, it is proposed that blood proteins reach the surface of the oocyte by an intercellular route, that they combine with some component of the brush border, and that they are transformed into yolk spheres by a process akin to pinocytosis.

Electron microscope studies on blue-green algae.

Abstract: Several species of blue-green algae were studied in thin sections with the electron microscope. Our electron micrographs confirm the view that the cell of blue-green algae is different and simpler in organization than the typical plant or animal cell. On the other hand, the general pattern of ultrastructure is the same as that found in bacteria and Streptomyces. The cell boundary is formed by a double membrane which consists of two typical unit membranes. Situated in between these membranes is the dense inner investment or wall which continues uninterrupted into the cross-walls. The cells always contain photosynthetic lamellae, nucleoplasm with DNA, small granules resembling ribosomes, and often also a number of larger granules of various sorts. The photosynthetic membranes either form the boundary of vesicles or flattened sacs, or, when the lumen of the vesicles disappears and the vesicular surfaces of the membranes zip together, they appear as lamellae made of two closely applied unit membranes. These vesicles or lamellae are disposed irregularly through the cell or arranged in parallel stacks of two or more. A thin layer of cytoplasm always separates the lamellae. The nucleoplasm is composed of masses of fine fibrils about 25 A thick and is either dispersed through the cell or concentrated in polymorphous reticular structures near the center of the cell. The improved resolution of the electron microscope makes it obvious that the terms "chromatoplasm" and "centroplasm" commonly used in the description of blue-green algae are really misleading. There are not different kinds of cytoplasm, but the cell consists of various structural (and functional) units like the ones mentioned above, which are arranged in the cell in a number of ways characteristic for each species or for different physiological or developmental states.

Centriole replication. A study of spermatogenesis in the snail Viviparus.

Abstract: This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO(4) and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mmicro in diameter and 330 mmicro long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are derived from the centromeres of degenerating chromosomes.

The formation of fibrils from collagen solutions. IV. Effect of mucopolysaccharides and nucleic acids: an electron microscope study.

Abstract: The kinetics of collagen reprecipitation from solutions of salt-extracted calf dermis in the presence of small amounts of mucopolysaccharide and nucleic acids (0.005 per cent in the final reaction mixture) has been reported by Wood (1960). The present paper is a parallel study using the same materials, and describes the electron microscopic (EM) morphology of the collagen precipitates replicated after 24 hours at room temperature. Satisfactory, uncontaminated EM preparations were obtained which showed that all the deposits were fibrous and bore the 640 A cross-banding characteristic of collagen except some narrow, background fibrils 200 to 1000 A wide precipitated in the presence of heparin. These exhibited fine striations about 220 A apart. Chondroitin sulfate greatly increased the rate of precipitation to give a deposit of low optical density consisting of narrow, rigid, discrete fibrils resembling fresh dermis. In contrast, heparin prevented macroscopic gelation, delayed precipitation, and only produced a scanty deposit of abnormal, short, wide, striated tactoids and compound fibers of varying length. The control preparations and the deposits formed in the presence of hyaluronic acid were intermediate between these two extremes. Delayed precipitation was associated with a coarser deposit and aggregation of the fibrils. A duplicate series of deposits precipitated in the presence of RNA and DNA, together with their controls, were examined after ½, 1, 1½, 3, 9, and 24 hours. One set employed an acetic extract of whole calf dermis and the other salt-extracted dermis. The presence of 0.005 per cent DNA in the reaction mixture markedly delayed collagen precipitation with the slow formation of abnormal, short, wide tactoids and compound fibers. RNA also interfered with the quantity and quality of the deposits which contained far less collagen resembling unfixed, normal, adult human dermis, than the controls at the corresponding time intervals. Comparison of the experiments employing whole calf dermis with those employing the salt-extracted material demonstrated that at every time interval in all the experiments the deposits were retarded when salt-extracted dermis was used. This suggests that the salt-soluble components of the dermis play a part in fiber formation.

The fine structure of nerve cell bodies and their myelin sheaths in the eighth nerve ganglion of the goldfish

Abstract: The eighth cranial nerve ganglion consists of bipolar nerve cell bodies each occupying part of an internodal segment. The perikaryal sheaths range from a single layer of Schwann cell cytoplasm on the smallest cells to typical thick compact myelin on the largest. On most perikarya, the sheath displays an intermediate form, consisting of multiple layers of Schwann cell cytoplasm (loose myelin), or of loose and compact myelin continuous with each other. Internodes beyond the one containing the cell body bear only compact myelin. In loose myelin the thickness of each layer of Schwann cell cytoplasm is about 100 A. It may be much greater (∼ 3000 A) particularly in the outermost layers of the sheath, or the cytoplasm may thin and even disappear with formation of a major dense line. The cytoplasmic layers are separated from each other by a light zone, 40 to 200 A wide, which in its broader portions may contain an intermediate line. Desmosomes sometimes occur between lamellae. In addition to the usual organelles, the perikaryal cytoplasm contains granular and membranous inclusions. Large cells covered by compact myelin have a consistently higher concentration of neurofilaments, and some of the largest cells, in addition, show a reduced concentration of ribosomes. The functional significance and possible origins of perikaryal myelin sheaths are discussed.

Studies on the content and organization of the respiratory enzymes of mitochondria

Abstract: (1) The mathematical calculations relating spectrophotometric data with the data of Allard et al. (4, 5) on mitochondrial counts, is presented. Such a calculation indicates that an "average mitochondrion" from rat liver would contain about 17,000 molecules of each cytochrome pigment. (2) Hematocrit determinations relating respiratory pigment content for mitochondria isolated from a variety of tissues have been presented, showing a fivefold variability depending upon the source of the mitochondria. (3) Speculations on the organization of the respiratory enzymes associated with the membrane structure of the mitochondria are discussed.

Functional analysis of swim-bladder muscles engaged in sound production of the toadfish

Abstract: A functional analysis of the striated swim-bladder muscles engaged in the sound production of the toadfish has been performed by simultaneous recording of muscle action potentials, mechanical effects, and sound. Experiments with electrical nerve stimulation were made on excised bladder, while decerebrate preparations were used for studies of reflex activation of bladders in situ. The muscle twitch in response to a single maximal nerve volley was found to be very fast. The average contraction time was 5 msec. with a range from 3 to 8 msec., the relaxation being somewhat slower. The analysis of muscle action potentials with surface electrodes showed that the activity of the muscle fibers running transversely to the long axis of the muscle was well synchronized both during artificial and reflex activation. With inserted metal microelectrodes monophasic potentials of 0.4 msec. rise time and 1.2 to 1.5 msec. total duration were recorded. The interval between peak of action potential and onset of contraction was only 0.5 msec. Microphonic recordings of the characteristic sound effect accompanying each contraction showed a high amplitude diphasic deflection during the early part of the contraction. During relaxation a similar but smaller deflection of opposite phase could sometimes be distinguished above the noise level. The output from the microphone was interpreted as a higher order derivative function of the muscle displacement. This interpretation was supported by complementary experiments on muscle sound in mammalian muscle. The dependence of the sound effects on the rate of muscle contraction was demonstrated by changing the temperature of the preparation and, in addition, by a special series of experiments with repeated stimulation at short intervals. Results obtained by varying the pressure within the bladder provided further evidence for the view that the sound initiated in the muscle is reinforced by bladder resonance. Analysis of spontaneous grunts confirmed the finding of a predominant sound frequency of about 100 per second, which was also found in reflexly evoked grunts. During these, muscle action potentials of the same rate as the dominant sound frequency were recorded, the activity being synchronous in the muscles on both sides. Some factors possibly contributing to rapid contraction are discussed.