REDUCTION OF HEATING ARTIFACTS IN THIN SECTIONS EXAMINED IN THE ELECTRON MICROSCOPE

Certain phenomena affecting contrast obtained from tissue sections with the electron microscope have been investigated and a technique is described for reducing destruction by the electron beam of fine details in sections. It has been concluded that loss of embedding material is slightly higher at exposed surfaces of sections than it is at surfaces covered by substrate film. Covering of both surfaces of sections with thin films of formvar, collodion, or carbon materially improves the general appearance, reduces distortion, and sometimes reduces loss of tissue mass from the section as result of exposure to the electron beam. This improvement is considered to result from the relatively high melting-point of the covering films which serve to eliminate or reduce surface-tension or other forces operating in methacrylate softened by the electron beam.     

A CYTOLOGICAL STUDY OF THE ALBUMIN-SECRETING CELLS OF THE HEN OVIDUCT

1. The electron and light microscope have been employed in a cytological study of the albumin-secreting cells of the hen oviduct and of fractions of this tissue obtained after homogenization and differential centrifugation. 2. These studies confirm the observation that in this tissue material corresponding to liver "microsomes" in amino acid-incorporating ability and ribonucleic acid content sediments in relatively low centrifugal fields. 3. The electron microscope studies suggest that the protein secretion of the gland is formed in intimate association with the ergastoplasm.     

THE PROBLEM OF DEMINERALISATION IN THIN SECTIONS OF FULLY CALCIFIED BONE

Thin sections of embryonic avian bone decalcify during preparation for electron microscopy, creating a false impression of mineral distribution. The results of the experiments reported herein show that viscous embedding materials do not penetrate compact formed bone, and so, in thin sections, the calcium apatite crystals may be leeched out by water, both in the collecting trough and in aqueous solutions of stains used to enhance tissue electron opacity. To prevent decalcification, a simple technique is described in which the aqueous fluids that come in contact with thin sections are saturated with respect to calcium and phosphate ions, thereby preventing solution of the bone mineral. The theoretical basis of this technique is briefly discussed.     

STUDIES ON THE PROBLEM OF PRESERVATION OF MYELIN SHEATH ULTRASTRUCTURE: EVALUATION OF FIXATION, DEHYDRATION, AND EMBEDDING TECHNIQUES

Currently accepted methods of tissue preparation for electron microscopy result in alterations of myelinated nerve fibers. In an attempt to minimize distortion of myelin, various fixation techniques, dehydration schedules, and embedding methods have been evaluated. It was found that the major damage to myelinated nerves occurs in the embedding procedure. A technique for embedding nerve tissue using the polyester Vestopal W is described which was found to result in improved preservation of myelin.     

STUDIES OF THE MITOCHONDRIAL ENERGY-TRANSFER SYSTEM OF BROWN ADIPOSE TISSUE

An investigation of the mechanisms of norepinephrine action and heat production in brown adipose tissue from newborn rabbits has been carried out. Data obtained with the use of biochemical techniques has been correlated with morphological data from electron microscopy. Norepinephrine was found to stimulate the respiration of brown fat in vitro. Inhibitors of glycolysis abolish this effect, whereas inhibitors of oxidative phosphorylation do not, at least not to the same extent. Brown fat is readily permeable to added Krebs cycle intermediates. Substrate level phosphorylation, but no electron transport-coupled phosphorylation, could be demonstrated in isolated mitochondria. It is suggested that the rate of fatty acid oxidation is limited by the availability of phosphate acceptor systems which break down ATP formed at the substrate level and thus provide ADP for further substrate level phosphorylation. The theory of respiratory control by the action of reesterification of fatty acids is discussed in the light of these findings. Under the electron microscope, brown fat mitochondria are characterized by their large size, tightly packed cristae, and by the different types of granules in the matrix. No elementary particles are seen when the mitochondria are examined by the negative-staining technique. The absence of electron transport-coupled phosphorylation together with the apparent absence of elementary particles seems to be of particular significance.     

ULTRASTRUCTURE OF AUXIN-INDUCED TUMORS OF THE COLEORHIZA-EPIBLAST OF WHEAT

When wheat is germinated in high concentrations of certain auxins, the coleorhiza-epiblast grows in an excessive and disorganized manner and resembles a callus. Wheat was germinated in 10⁻³ M indoleacetic acid or in water. There was greater net synthesis of DNA, RNA, and protein in the tumor tissue than in control tissue. Control and tumor tissue was fixed for electron microscopy 1, 2, 3, and 4 days after sowing. In contrast to the controls, many more lipoidal bodies appeared in 1-day-old, auxin-treated tissue and thereafter diminished in number. In 2- and 3-day-old tumor tissue, small membraneous fragments were prominent but disappeared in older tissue. With time, the following changes became progressively more pronounced: cytoplasmic vacuolation, appearance of myelin figures, polyribosome configurations and extensive profiles of rough endoplasmic reticulum, and sloughing of cytoplasmic contents into and accumulation of electron-dense material in vacuoles. Possible factors in susceptibility of certain tissues to tumor formation are discussed.     

LONG-TERM ORGAN CULTURE OF THE SALAMANDER HEART

Beating salamander hearts were maintained in tissue culture for periods ranging from 1 to 6 months. After 1, 3, or 6 months of culture, six hearts, along with six control hearts, were fixed for electron microscopy. In control tissue, the sarcoplasmic reticulum usually demonstrated the normal pattern of paired, linearly arranged membranes, although in some cases, the reticulum showed a variation from these membranes to a series of small vesicles. There was no evidence of a T-system of tubules in any of the material examined. Desmosome-Z band complexes were observed in almost all sections of both control and experimental material. A possible role of these complexes in the excitation-contraction mechanism is discussed. In 3 month cultured material, alterations in normal myofibrillar pattern occurred. Small segments of myofibrils branched from one Z band to join the Z band of an adjacent myofibril, or appeared to be fraying out into the sarcoplasm. In 6 month cultured material, myofibrils were fragmented into short segments from which myofilaments frayed out into the sarcoplasm. This filamentous material may be remnants of myofilaments. Despite the morphological changes in myofibrils, the heart pulsation rate, established at the beginning, was maintained throughout the culture period. It is suggested that the alterations, observed in the experimental material, occurred in elements not essential for heart beat maintenance, or that these alterations have not yet progressed to a critical point of affecting the heart beat.     

SOME EFFECTS OF THE MICROTOME KNIFE AND ELECTRON BEAM ON METHACRYLATE-EMBEDDED THIN SECTIONS

A technique for the examination of specimens at low electron beam intensity has been presented. Sections micrographed with this technique showed numerous knife scratches and frequently contained bands running parallel to the knife edge. Banding with an average spacing of 0.2 µ appeared to result from periodic distortion produced by impact of the knife. At the beam intensities customarily employed, differential sublimation and probably flow of the methacrylate resulted in obliteration of the bands and all but the deepest knife scratches. In addition, changes in the size, shape, and orientation of certain structures were noted. Artifacts resulting from incineration or sublimation of tissue components fixed in formalin were illustrated, and the suggestion was made that such instability to the electron beam accounted in part for the differences observed in osmium- and formalin-fixed tissues. The deformation revealed in serial sections was discussed, and it was pointed out that shortening in the axis perpendicular to the knife edge was associated with elongation in the axis parallel to the cutting edge, the elongation usually occurring locally without change in the width of the section. It was noted that the material causing contamination of the surface of sections during examination exhibited no structure but caused progressive loss of contrast.     

Embedding Plant Tissue with Plastic Using High Pressure: A New Method for Light and Electron Microscopy

An embedding technique has been developed to overcome difficulties that confront light and electron microscopists working with so-called “hard-to-embed” plant tissue. The method was originally described for freeze-dried material. It uses a modified Quickfit Rotaflo Valve and low heat to generate high pressure to aid in the infiltration and embedding of tissue with propylene oxide and plastic. The technique is not too cumbersome and requires 6 days from the dehydration step to the end of the polymerization process. Thick sections (1-2 μm) obtained from material prepared by this method stain readily with toluidine blue, and thin sections for the electron microscope stain satisfactorily following standard treatment with uranyl acetate and lead citrate. The thin sections are stable under the beam of the electron microscope. Results indicate that the quality of tissue preservation with this high pressure embedding technique is as good as that observed using standard embedding methods for electron microscopy.     

MORPHOGENESIS OF THE COLLAGENOUS STROMA IN THE CHICK CORNEA

The embryonic chick corneal epithelium produces a highly structured acellular matrix beneath its basal surface during early development. This matrix, which contains collagen, serves as a morphogenetic template for subsequent stromal development in that the three-dimensional architecture of the adult corneal stroma is initially established, in miniature, in this epithelially derived connective tissue. Examination of the early corneal epithelium and matrix in both the light and electron microscope suggests that self assembly of the matrix may be one of several important factors in the morphogenesis of this early connective tissue.     

STUDIES ON THE BIOSYNTHESIS OF COLLAGEN : I. THE GROWTH OF FOWL OSTEOBLASTS AND THE FORMATION OF COLLAGEN IN TISSUE CULTURE

1. A tissue culture method was devised in which suspensions of osteoblasts, obtained directly from frontal bones of fowl embryos, were grown in a fluid, fibrin-free medium. 2. Maximum growth of the tissue, as measured by dry weight, with the formation of collagen protein, based on the estimation of hydroxyproline, was obtained in periods of up to 6 days. 3. Appreciable amounts of protein-bound hydroxyproline were formed during the first 24 hour growth period, but electron microscopy of portions of the same cultures failed to demonstrate the presence of any typical collagen fibrils. 4. The subsequent formation of many characteristic collagen fibrils was not associated with a significant rise in the mean hydroxyproline content of the tissue. 5. The cytoplasmic granules of the osteoblasts stained intensely with the P.A.S. technique when the collagen fibrils were being formed. 6. It is suggested that collagen-forming cells synthesise and secrete a hydroxyproline-rich precursor of protein or large peptide nature, which subsequently becomes directly transformed into typical collagen fibrils.     

THE FINE STRUCTURE AND DEVELOPMENT OF THE COMPANION CELL OF THE PHLOEM OF ACER PSEUDOPLATANUS

At maturity the companion cell of the phloem of the sycamore Acer pseudoplatanus has a large nucleus, simple plastids closely sheathed with rough endoplasmic reticulum, and numerous mitochondria. The cytoplasm contains numerous ribosomes, resulting in a very electron-opaque cytoplasm after permanganate fixation. Bodies similar to the spherosomes of Frey-Wyssling et al. (4) are collected in clusters and these also contain bodies of an unidentified nature similar to those found by Buttrose (1) in the aleurone cells of the wheat grain. The pores through the wall between the companion cell and sieve tube are complex and develop from a single plasmodesma. Eight to fifteen plasmodesmata on the companion cell side communicate individually with a cavity in the centre of the wall which is linked to the sieve tube by a single pore about twice the diameter of an individual plasmodesma. This pore is lined with material of an electron opacity equivalent to that of material bounding the sieve plate pores. The development of the cell organelles, the possible role played in the phloem tissue by the companion cell, and the function of the complex pores contained in its wall are discussed.     

STRUCTURE AND DEVELOPMENT OF SIEVE ELEMENTS IN THE LEAF OF ISOETES MURICATA

Leaf tissue of Isoetes muricata Dur. was fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. The very young sieve elements can be distinguished from contiguous parenchyma cells by their distinctive plastids and the presence of crystalline and fibrillar proteinaceous material in dilated cisternae of the rough ER. During differentiation, the portions of ER enclosing this proteinaceous substance become smooth surfaced and migrate to the cell wall. Along the way they apparently form multivesicular bodies which then fuse with the plasmalemma, discharging their contents to the outside. At maturity, the sieve element contains an elongate nucleus, which consists of dense chromatin material, and remnants of the nuclear envelope. In addition, the mature sieve element is lined by a plasmalemma and a parietal, anastomosing network of smooth ER. Both plastids and mitochondria are present. P-protein is lacking at all stages of development. Tonoplasts are. not discernible in mature sieve elements. The end walls of mature sieve elements contain either plasmodesmata or sieve pores or both, but only plasmodesmata occur in the lateral walls.     

白菜细胞核雄性不育花药的细胞化学观察

对一种由一对隐性基因控制的白菜细胞核雄性不育和可育株的花药进行了细胞学和组织化学研究。种子播种后,有1/4植株为不育株,其余的为可育株。通过对不育株和可育株花药发育的细胞学观察,确认不育花粉的败育发生在小孢子发育时期。用组织化学的方法研究了可育株和不育株花药发育过程中的多糖和脂类的分布动态,发现在减数分裂前,可育花药和不育花药的药隔细胞中都储藏了大量的淀粉粒。二者的差异仅是不育花药的绒毡层细胞液泡化明显。在减数分裂后的小孢子发育时期,可育花药的绒毡层细胞具有将药隔细胞中的淀粉粒多糖吸收并转化成脂类的功能,小孢子及以后的二胞花粉中也积累了大量的脂类储藏物质在不育花药中,虽然减数分裂后药隔细胞中的淀粉粒也都消失,但绒毡层细胞中的脂类物质相比很少,同时绒毡层细胞显示了明显的多糖反应,表明不育花药的绒毡层细胞将糖类转化为脂类的功能受阻。在小孢子的表面有些脂类物质,但在细胞质中却没有脂类积累。这一结果暗示在该种白菜细胞核雄性不育株中,由于花药绒毡层细胞转换多糖为脂类的功能失常,导致了小孢子的败育。     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF THIN SECTIONS: THE GOLGI ZONE AS A SITE OF PROTEIN CONCENTRATION IN PANCREATIC ACINAR CELLS

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections (~ 600 A) of OsO₄-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4°C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H³ injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.     

THE FINE STRUCTURE OF THE MID-BODY OF THE RAT ERYTHROBLAST

The development of the mid-body has been studied in mitotic erythroblasts of the rat bone marrow by means of thin sections examined with the electron microscope. A differentiated region on the continuous spindle fibers, consisting of a localized increase in density, is observed at the equatorial plane. The mid-body seems to develop by the aggregation of such denser lengths of spindle fiber. Its appearance precedes that of the cleavage furrow. A plate-like arrangement of fibrillary material lies transversely across the telophase intercellular bridge. Later, this material becomes amorphous and assumes the form of a dense ring closely applied to a ridge in the plasma membrane encircling the middle of the bridge. Although the mid-body forms in association with the spindle fibers, it is a structurally distinct part, and the changes which it undergoes are not shared by the rest of the bundle of continuous fibers.     

THE INTERFACE BETWEEN COTTON TISSUE AND A PENETRATING NOBLE METAL PROBE

A passive system that may be useful for measuring changes of the redox potential of in vivo, extracellular plant tissue has been developed within the past few years. The technique involves the placement of a small (250 μm diam) noble metal probe into the anatomical region of interest and subsequent monitoring of the time variation in the measured electrode potential of this probe. The result of this measuring technique has been termed an electrophytogram. The observed voltage fluctuations may be empirically correlated with several observable plant growth phenomena. However, elucidation of the physiological basis for electrophytograms must involve the proper anatomical interpretation of the position of the probe relative to the plant tissue. This paper reports the results of freeze-fracture electron microscopic examination of the probe/tissue interface after wound healing has occurred. Electron micrographs clearly show cell wall material, appressed directly against the probe. These results indicate that the electrophytogram system provides a method for monitoring the electrochemical status of the cell wall space. Insertion of the probe into mature, fully elongated tissue appears to cause little physical damage to non-xylem tissue organization beyond the adjacent cell layers and virtually no damage to the xylem region.     

ULTRASTRUCTURE OF THE PSEUDOCAPILLITIUM AND SPORES OF THE MYXOMYCETE LYCOGALA EPIDENDRUM. LICEALES

The pseudocapillitium and spores of L. epidendrum were studied by transmission (TEM) and scanning electron microscopy (SEM). The SEM reveals that the pseudocapillitial surface is covered by bands of “wartlike” processes that alternate with non-ornamented regions. Otherwise, the pseudocapillitium is a hollow structure composed of three regions. The outer region is thin, electron dense and continuous with many irregular processes. Internal to this area is an amorphous region containing scattered electron dense material. The innermost region of the pseudocapillitium is thin, inconspicuous and usually electron dense. L. epidendrum possesses spores that are covered by a surface reticulum consisting of polygonal areas which are continuous with the outermost spore layer. The outer spore layer is thin and electron dense. The inner spore layer is an electron transparent region that contains granular or fibrillar components. Sections of spores showed a dense cytoplasm possessing most of the usual organelles along with microtubules and microbodies.