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Striated muscle fibers from the body and tail myotomes of a fish, the black Mollie, have been examined with particular attention to the sarcoplasmic reticulum (SR) and transverse tubular (or T) system. The material was fixed in osmium tetroxide and in glutaraldehyde, and the images provided by the two kinds of fixatives were compared. Glutaraldehyde fixes a fine structure that is broadly comparable with that preserved by osmium tetroxide alone but differs in some significant details. Especially significant improvements were obtained in the preservation of the T system, that is, the system of small tubules that pervades the fiber at every Z line or A-I junction level. As a result of this improved glutaraldehyde fixation, the T system is now clearly defined as an entity of fine structure distinct from the SR but uniquely associated with the SR and myofibrils. Glutaraldehyde fixation also reveals that the T system is a sarcolemmal derivative that retains its continuity with the sarcolemma and limits a space that is in direct communication with the extracellular environment. These structural features favor the conclusion that the T system plays a prominent role in the fast intracellular conduction of the excitatory impulse. The preservation of other elements of muscle fine structure, including the myofibrils, seems for reasons discussed, to be substantially improved by glutaraldehyde.

The macronucleus in Tokophrya infusionum is composed of numerous Feulgen-positive chromatin bodies (about 0.5 micro in diameter) which appear in thin sections as a dense spongework, homogeneous throughout. The same appearance characterizes metaphase chromosomes of higher forms. Some chromatin bodies of the macronucleus were found to possess a highly organized structure in certain old organisms. This structure appears in cross-sections as a honeycomb and in longitudinal sections as parallel lines about 120 A in diameter evenly spaced (about 230 A). As far as is known this is the first time a regular structure has been found in bodies of chromosomal character at the dimensional level presently explored by electron microscopy. The demonstration that OsO(4) can preserve order in chromatin material is another significant aspect of these findings.

Pigment epithelial cells of the frog's retina have been examined by methods of electron microscopy with special attention focused on the fine structure of the endoplasmic reticulum and the myeloid bodies. These cells, as reported previously, send apical prolongations into the spaces between the rod outer segments, and within these extensions, pigment migrates in response to light stimulation. The cytoplasm of these cells is filled with a compact lattice of membrane-limited tubules, the surfaces of which are smooth or particle-free. In this respect, the endoplasmic reticulum here resembles that encountered in cells which produce lipid-rich secretions. The myeloid bodies comprise paired membranes arranged in stacks shaped like biconvex lenses. At their margins the membranes are continuous with elements of the ER and in consequence of this the myeloid body is referred to as a differentiation of the reticulum. The paired membranes resemble in their thickness and spacings those which make up the outer segments; they are therefore regarded as intracellular photoreceptors of possible significance in the activation of pigment migration and other physiologic functions of these cells. The fuscin granules are enclosed in membranes which are also continuous with those of the ER. The granules seem to move independently of the prolongations in which they are contained. The report also describes the fine structure of the terminal bar apparatus, the fibrous layer intervening between the epithelium and the choroid blood vessels, and comments on the functions of the organelles depicted.

Several types of striated muscle have been examined by the technics of electron microscopy and the findings in myotome fibers of Amblystoma larvae, the sartorius, and cardiac muscle of the rat are reported on in some detail. Particular attention has been given to structural components of the interfibrillar sarcoplasm and most especially to a finely divided, vacuolar system known as the sarcoplasmic reticulum. This consists of membrane-limited vesicles, tubules, and cisternae associated in a continuous reticular structure which forms lace-like sleeves around the myofibrils. It shows a definable organization which repeats with each sarcomere of the fiber so that the entire system is segmented in phase with the striations of the associated myofibrils. Details of these repetitive patterns are presented diagrammatically in Text-figs. 1, 2, and 3 on pages 279, 283, and 288 respectively. The system is continuous across the fiber at the H band level and largely discontinuous longitudinally because of interruptions in the structure at the I and Z band levels. The structure of the system relates it to the endoplasmic reticulum of other cell types. The precise morphological relation of the reticulum to the myofibrils, with specializations opposite the different bands, prompts the supposition that the system is functionally important in muscle contraction. In this regard it is proposed that the membrane limiting the system is polarized like the sarcolemma and that the corresponding potential difference is utilized in the intracellular distribution of the excitatory impulse.

Skeletal myofibrils isolated either by tryptic digestion at 0 degrees C. or by a colloid mill and suspended in buffer solution (pH 7.0, micro; 0.154) containing 20 per cent glycerin and 0.0025 M adenosinetriphosphate at -5 degrees C. contracted slowly and progressively when the temperature was raised above 0 degrees C. Formalin fixation halted this contraction. With the aid of these procedures myofibrils in progressive stages of contraction were then studied with the electron microscope. Electron micrographs showed that uncontracted fibrils isolated by the colloid mill were structurally similar to those described by other workers. Treatment of fibrils with trypsin removed the Z bands and disorganized the I bands. This enzymatic modification of structure did not impair the contractile response. The principal structural changes during contraction consisted of a migration of dense material from the A band into the A-I junction or the Z band, a gradual increase in width of the fibril, a gradual decrease in length of sarcomeres, an apparent increase in the mean diameter of filaments, and a disorientation of these latter from their parallel arrangement.

This investigation has sought to determine the relation between collagen fiber and fibroblast during fibrogenesis. Toward this end the surfaces of chick fibroblasts grown under in vitro conditions have been examined with the electron microscope after fixation in OsO(4). Supplementary information has been obtained from thin sections of fibroblasts fixed in situ during phases of fiber production. The evidence provided by these studies and by various conditions of the experiments indicates that the unit fibrils of collagen form in close association with the cell surface. They were never observed within the cell. When these unit fibrils form in bundles it appears as though templates of some nature, possibly coinciding with stress fibers within the cell cortex, influence the polymerization of the fibrils out of material available at the cell surface. From here the fibrils and bundles of them are shed into the intercellular spaces and there grow to limited diameters by accretion of materials from the general milieu.

Cells of onion and garlic root tips were examined under the electron and phase contrast microscopes after fixation in KMnO(4). Special attention was focused on the distribution and behavior of the endoplasmic reticulum (ER) during the several phases of mitosis. Slender profiles, recognized as sections through thin lamellar units of the ER (most prominent in KMnO(4)-fixed material), are distributed more or less uniformly in the cytoplasm of interphase cells and show occasional continuity with the nuclear envelope. In late prophase the nuclear envelope breaks down and its remnants plus cytoplasmic elements of the ER, which are morphologically identical, surround the spindle in a zone from which mitochondria, etc., are excluded. During metaphase these ER elements persist and concentrate as two separate systems in the polar caps or zones of the spindle. At about this same time they begin to proliferate and to invade the ends of the spindle. The invading lamellar units form drape-like partitions between the anaphase chromosomes. In late anaphase, their advancing margins reach the middle zone of the spindle and begin to fray out. Finally, in telophase, while elements of the ER in the poles of the spindle coalesce around the chromosomes to form the new envelope, the advancing edges of those in the middle zone reticulate at the level of the equator to form a close lattice of tubular elements. Within this, which is identified as the phragmoplast, the earliest signs of the cell plate appear in the form of small vesicles. These subsequently grow and fuse to complete the separation of the two protoplasts. Other morphological units apparently participating in mitosis are described. Speculation is provided on the equal division or not of the nuclear envelope and the contribution the envelope fragments make to the ER of the new cell.

Electron microscopy of thin sections of muscle fibers in myotomes of Amblystoma larvae has revealed the presence of a complex, membrane-limited system of canaliculi and vesicles which form a lace-like reticulum around and among the myofibrils. This seems to correspond to the sarcoplasmic reticulum of the earlier light microscopists and the endoplasmic reticulum of other cell types. The elements constituting the reticulum are disposed in a pattern which bears a constant relation to the bands of the adjacent myofibrils and is therefore repeated in each sarcomere. At the H band the system is transversely continuous but not so at other levels. Longitudinally continuity is interrupted at the Z bands where large vesicles belonging to adjacent sarcomere segments of the system face off on opposite sides of the band. The opposing faces of these vesicles are flat and separated by a space of more or less constant width, in which are located small, finger-shaped vesicles. In view of these and other close structural relationships with the myofibrils it seems appropriate to assign to the system a role in the conduction of the excitatory impulse.

The course of events in the solvation and reconstitution of collagen obtained from rat tail tendon is described as seen with the electron microscope. Under the influence of 0.01 per cent acetic acid the collagen fibers swell and dissociate into submicroscopic filaments, the smallest of which are probably beyond the resolution of the electron microscope. These filaments can be made to reconstitute into fibers either by the addition of neutral salts to the acid solution or by raising the pH. The structural form of the resulting fibers is influenced by the concentration of the salt and by the pH of the solution employed. Saline-concentrations around 1 per cent and pH's ranging from 4.8 to 6.8 lead to the formation of needle-shaped crystals, or tactoids, showing the striated structure characteristic of collagen. Saline concentrations outside of this range (0.5 per cent and 5.0 per cent) lead to the formation of long fibrils without evidence of striations. pH's on the alkaline side of 6.8 bring about the formation of long slender fibers. Some of the possible reasons for these different fiber forms are discussed. Apparently fibers are formed in vitro by the lateral and longitudinal association of the filaments seen in the original solutions. Some of the fibers thus formed may in turn associate laterally and longitudinally to form the larger fibers. The formation of the needle-shaped crystals appears to be an orderly process since it leads to the formation of a periodicity in the fibers. The striations in the smaller fibers are uniform and regularly spaced at around 210 A. With continued growth of these fibers there is a simultaneous development of a more obvious and precise banding. It is evident that two out of each group of three regularly spaced striae are more prominent. This produces the macroperiod of collagen measuring around 640 A in length.

The cytoplasmic ground substance of animal tissue cells grown in vitro has been found by electron microscopy to contain, as a part of its submicroscopic structure, a complex reticulum of strands, to be referred to as the endoplasmic reticulum. It has been found in all types of cells extensively studied. The components of this reticular system vary considerably in size and form, apparently in some relation to physiological changes in the cell. Thus in one cell of a culture colony it may be finely divided into strands or canaliculi, 50 to 100 mmicro in diameter, whereas in an adjacent cell of the same type the components of the reticulum may be relatively coarse, 600 mmicro in diameter, and vesiculated. The membrane, which can be shown to limit the system and separate it from the rest of the ground substance, is similar in thickness to the plasma membrane surrounding the cell. Photomicrographs of living cells taken by phase contrast and dark field microscopy define a structure of similar form and indicate that the reticulum of the electron microscope image has its equivalent in the living unit. Where its component units are sufficiently large, a structure of identical form can be resolved by light microscopy in cells stained with hematoxylin or with toluidine blue. This indicated that the endoplasmic reticulum is to be identified with the basophilic or chromophilic component (the ergastoplasm) of the cytoplasm and that such properties of this component as have been determined by cytochemical methods, such as a high RNA content, may be assigned to this "submicroscopic" system.

The role of the cells in the fabrication of a connective tissue matrix, and the structural modifications which accompany cytodifferentiation have been investigated in developing epiphyseal cartilage of fetal rat by means of electron microscopy. Differentiation of the prechondral mesenchymal cells to chondroblasts is marked by the acquisition of an extensive endoplasmic reticulum, enlargement and concentration of the Golgi apparatus, the appearance of membrane-bounded cytoplasmic inclusions, and the formation of specialized foci of increased density in the cell cortex. These modifications are related to the secretion of the cartilage matrix. The matrix of young hyaline cartilage consists of groups of relatively short, straight, banded collagen fibrils of 10 to 20 mmicro and a dense granular component embedded in an amorphous ground substance of moderate electron density. It is postulated that the first phase of fibrillogenesis takes place at the cell cortex in dense bands or striae within the ectoplasm subjacent to the cell membrane. These can be resolved into sheaves of "primary" fibrils of about 7 to 10 mmicro. They are supposedly shed (by excortication) into the matrix space between the separating chondroblasts, where they may serve as "cores" of the definitive matrix fibrils. The diameter of the fibrils may subsequently increase up to threefold, presumably by incorporation of "soluble" or tropocollagen units from the ground substance. The chondroblast also discharges into the matrix the electrondense amorphous or granular contents of vesicles derived from the Golgi apparatus, and the mixed contents of large vacuoles or blebs bounded by distinctive double membranes. Small vesicles with amorphous homogeneous contents of moderate density are expelled in toto from the chondroblasts. In their subsequent evolution to chondrocytes, both nucleus and cytoplasm of the chondroblasts undergo striking condensation. Those moving toward the osteogenic plate accumulate increasingly large stores of glycogen. In the chondrocyte, the enlarged fused Golgi vesicles with dense contents, massed in the juxtanuclear zone, are the most prominent feature of the cytoplasm. Many of these make their way to the surface to discharge their contents. The hypertrophied chondrocytes of the epiphyseal plate ultimately yield up their entire contents to the matrix.

A series of representative cell types including avian fibroblasts, and macrophages; rabbit mesothelia, endothelia, and nephron epithelia; and rat glandular epithelia (parotid) were studied comparatively in vitro and in situ with the electron microscope. Cells in vitro were examined in whole mounts and in sections whereas cells in situ were observed exclusively in sections. It was found that an endoplasmic reticulum similar to that previously described in cultured material is present in situ in all cell types examined. Modifications in its appearance introduced by the sectioning technique were discussed and explained. The observations showed in addition that the endoplasmic reticulum is a network of cavities which may enlarge into relatively vast, flattened vesicles here described as cisternae.

Yolk proteins are thought to enter certain eggs by a process akin to micropinocytosis but the detailed mechanism has not been previously depicted. In this study the formation of protein yolk was investigated in the mosquito Aedes aegypti L. Ovaries were fixed in phosphate-buffered osmium tetroxide, for electron microscopy, before and at intervals after a meal of blood. The deposition of protein yolk in the oocyte was correlated with a 15-fold increase in 140 mmicro pit-like depressions on the oocyte surface. These pits form by invagination of the oocyte cell membrane. They have a 20 mmicro bristle coat on their convex cytoplasmic side. They also show a layer of protein on their concave extracellular side which we propose accumulates by selective adsorption from the extraoocyte space. The pits, by pinching off from the cell membrane become bristle-coated vesicles which carry the adsorbed protein into the oocyte. These vesicles lose the coat and then fuse to form small crystalline yolk droplets, which subsequently coalesce to form the large proteid yolk bodies of the mature oocyte. Preliminary radioautographs, and certain morphological features of the fat body, ovary, and midgut, suggest that the midgut is the principal site of yolk protein synthesis in the mosquito.

The electron microscope was used to study the structure and three dimensional relationships of the components of the body cortex in thin sections of Paramecium multimicronucleatum. Micrographs of sections show that the cortex is covered externally by two closely apposed membranes (together approximately 250 A thick) constituting the pellicle. Beneath the pellicle the surface of the animal is molded into ridges that form a polygonal ridgework with depressed centers. It is these ridges that give the surface of the organism its characteristic configuration and correspond to the outer fibrillar system of the light microscope image. The outer ends of the trichocysts with their hood-shaped caps are located in the centers of the anterior and posterior ridges of each polygon. The cilia extend singly from the depressed centers of the surface polygons. Each cilium shows two axial filaments with 9 peripheral and parallel filaments embedded in a matrix and the whole surrouned by a thin ciliary membrane. The 9 peripheral filaments are double and these are evenly spaced in a circle around the central pair. The ciliary membrane is continuous with the outer member of the pellicular membrane, whereas the plasma membrane is continuous with the inner member of the pellicular membrane. At the level of the plasma membrane the proximal end of the cilium is continuous with its tube-shaped basal body or kinetosome. The peripheral filaments of the cilium, together with the material of cortical matrix which tends to condense around them, form the sheath of the basal body. The kinetodesma connecting the ciliary kinetosomes (inner fibrillar system of the light microscopist) is composed of a number of discrete fibrils which overlap in a shingle-like fashion. Each striated kinetosomal fibril originates from a ciliary kinetosome and runs parallel to other kinetosomal fibrils arising from posterior kinetosomes of a particular meridional array. Sections at the level of the ciliary kinetosomes reveal an additional fiber system, the infraciliary lattice system, which is separate and distinct from the kinetodesmal system. This system consists of a fibrous network of irregular polygons and runs roughly parallel to the surface of the animal. Mitochondria have a fine structure similar in general features to that described for a number of mammalian cell types, but different in certain details. The structures corresponding to cristae mitochondriales appear as finger-like projections or microvilli extending into the matrix of the organelle from the inner membrane of the paired mitochondrial membrane. The cortical cytoplasm contains also a particulate component and a system of vesicles respectively comparable to the nucleoprotein particles and to the endoplasmic reticulum described in various metazoan cell types. An accessory kinetosome has been observed in oblique sections of a number of non-dividing specimens slightly removed from the ciliary kinetosome and on the same meridional line as the cilia and trichocysts. Its position corresponds to the location of the kinetosome of the newly formed cilium in animals selected as being in the approaching fission stage of the life cycle.

Fibroblasts active in collagen production show a rich development of the endoplasmic reticulum (ER) and an enlarged Golgi complex, both characteristic of cells engaged in protein synthesis. The relatively quiescent fibrocyte, on the other hand, is deficient in these same cytoplasmic systems. When fibroblasts (or chondroblasts) are provided with tritiated (H(3)) proline, the label shows, by autoradiography, incorporation first into materials (collagen) in the cisternae of the ER, transfer thence in time to the Golgi, and eventual secretion into the extracellular environment. Sulfur(25) incorporation into chondroitin sulfate appears to involve only structural elements of the Golgi complex. There is increasing evidence of intimate fibroblast (or cell) involvement in the initiation and orientation of unit collagen fibrils. This question is reexamined in relation to the development of the prominent basement lamella of young adult lampreys.

The observed sequences in the formation of clots from purified bovine fibrinogen and thrombin are described. Under the conditions of these experiments, it appears that fibrinogen molecules are polymerized by the action of thrombin to form needle-shaped, crystal-like protofibrils which then become aligned into fiber strands by lateral association. The integrity of the unit fibrils is maintained within the strand. A model of the fibrinogen molecule is proposed which may satisfy the reported physical constants, data from x-ray diffraction studies, and observations made upon electron micrographs.

This paper reports on the fine structure of rat oocytes at stages before ovulation, during maturation, fertilization, and early cleavage. The study includes parallel observations on light and electron microscope preparations with attempted correlations. The follicular cells of the ovarian egg are described as sending long processes through the zona pellucida to the egg surface where they mingle with thin projections from the egg itself. No open communication between follicle cell cytoplasm and egg cytoplasm was observed. During maturation and fertilization both types of processes are withdrawn from the zona. The germinal vesicle and later the pronuclei of the fertilized egg are characterized by numerous large nucleoli. These have the form of thick walled vesicles with diameters as great as 8 to 10 micro. The wall is dense in the EM image and appears to consist in part of small granules. The cytoplasm shows several inclusions including mitochondria of usual form and a Golgi component which has the typical fine structure and the distribution described by earlier light studies. Small dense particles, presumably RNP particles, are distributed throughout the cytoplasmic matrix and show no preference for membranes. The endoplasmic reticulum of the oocyte is represented by a scattering only of vesicles, but begins a more extensive and elaborate development with the onset of segmentation. One inclusion of the ooplasm, similar in size to mitochondria, receives special attention. It is a vesicular structure, containing a large number of small vesicles (10 to 50 mmicro in diameter) and frequently a central density or nucleoid. They are referred to as multivesicular bodies. Such bodies are found in small number in the ovarian egg, but increase greatly in number during maturation and fertilization. It appears from the micrographs of eggs in these latter stages that these vesicular bodies break down and liberate their content of small vesicles to the surrounding ooplasm. Comments are provided on the apparent significance of the various observations.