An Electron Microscope Study of the Salamander Thyroid during Hormonal Stimulation

Cytological changes in thyroid glands following administration of thyroid-stimulating hormone (TSH), were studied in adult salamanders, Ambystoma tigrinum, Triturus torosus, and Triturus viridescens by electron and light microscopy. Thyroids from untreated salamanders contained large follicles, faintly basophilic colloid, low follicle cells with flattened nuclei, and scant, slightly basophilic cytoplasm. After TSH administration the cell height and nuclear volume increased. Cytoplasmic basophilia was markedly increased and follicle lumina were reduced. In electron micrographs, stacks of ergastoplasmic lamellae appeared near the nucleus occasionally in contact with the nuclear membrane. In more advanced stages of stimulation, lamellar arrays were largely replaced by small disoriented vesicles and larger vacuoles containing colloid-like material. Sections of obliquely oriented ergastoplasmic membranes contained rows of extremely fine particles. Microvilli increased in size and number and Golgi structures became more extensive. Homogeneous osmiophilic droplets increased in size and abundance. Some of the smaller droplets were seen associated with the Golgi zone. Droplets similar in size and density frequently contained closely packed, whorled lamellae. Mitochondria showed no structural changes but occurred in aggregates interposed between the nucleus and highly folded portions of the basal cell membrane.     

Cytochemical Localization of Peroxidase in Plant Cells

Plant cells were stained for peroxidase by exposing them to a mixture of p-phenylene diamine and hydrogen peroxide. Peroxidase activity is shown to be associated with chromosomes, nucleoli, cell walls and cytoplasm. Possible physiological roles of peroxidase bound to different structures are discussed.     

基于抗坏血酸过氧化酶的电镜成像和活细胞邻近标记方法的应用进展

Alice Ting实验室开发的抗坏血酸过氧化物酶(engineered ascorbate peroxidase,APEX),相对于经典的辣根过氧化物酶(horse radish peroxidase,HRP),其酶活性不再受细胞内蛋白质定位的影响,可以在几乎所有的亚细胞区域保持活性,这使其在研究亚细胞尺度以及活细胞水平生物学问题时极具优势.目前,基于APEX的二氨基联苯胺(diaminobenzidine,DAB)染色标记技术已经成功地实现对全细胞、亚细胞器和蛋白质水平的电镜成像.同时,与质谱技术结合,基于APEX的活细胞生物素邻近标记方法也极大地推动了亚细胞器蛋白质组学,以及目标蛋白在特定时空条件下邻近蛋白质组学的研究发展.本文将从以上两个方面阐述APEX技术的基本原理及最新应用进展,并讨论和展望其在实际应用中存在的局限性和挑战.     

扫描电镜技术在纤维开发研究中的应用

探讨了纤维扫描电镜样品制备技术,并用扫描电镜观察了木浆纤维、回用纸浆纤维和微晶纤维素的内部结构变化.结果表明:新木浆打浆后,纤维内部结构保存良好,可保持纸浆纤维的柔韧性和强度;回用纸浆纤维打浆后,纤维内部结构受到损伤,其程度随打浆次数的增加而加剧,降低了纸浆纤维的柔韧性和强度;化学水解和机械粉碎可使微晶纤维素的内部结构出现裂隙,有利于提高其与抗菌素等物质之间的吸附能力.     

Electron Microscopic Cytochemical Studies on the Glycoproteins in Cellular Membranes of Winter Wheat Seedlings with Concanavalin A-Horseradish Peroxidase Method

The localization and distribution of the glycoproteins in cellular membranes of strong cold hardy winter wheat (Triticum aestivum L. cv. Yanda 1817) seedlings were studied employing the modified cytochemical method using Concanavalin A-Horseradish peroxidase conjugates. The results obtained showed that the reaction products of Horseradish peroxidase activity which indicated the presence of glycoproteins appeared as individual particles distributed at plasmalemma, endoplasmic reticulmn, nuclear envelope and tonoplast in the leaflet cells of the seedlings grown under optimum temperature 25℃ day/20℃ night. After cold acclimation of the seedlings at low temperature during late autumn and early winter, the quantity of glycoproteins distributed in endoplasmic reticulum and nuclear envelope was significantly increased, and glycoproteins were transported into almost all of the plasmodesmata. At some sites of mitochondria and plastides newly formed glycoproteins were observed. However, no distribution of glycoproteins was for,nd in Golgi bodies during the whole observations. The role of glycoproteins for the development and stability of cold resistance in plant through blocking up the plasmodesma passage in the overwintering period of winter wheat seedlings, and the possible differences of the synthetic site and transportation pathway of glycoproteins between animal and plant systems were discussed.     

CYTOPLASMIC LABEL FOLLOWING TRITIATED THYMIDINE TREATMENT OF ALLIUM CEPA L. ROOTS : Cytochemical and Electron Microscope Study

Tritiated thymidine routinely labels onion root cytoplasm during most of the cell cycle. One-third of this label could be cytochemically identified as DNA. The balance of the label was not RNA or a lipid, or attributable to labeled impurities in thymidine-³H. In electron microscope radioautographs one-third of the cytoplasmic silver grains was over organelles, presumably mitochondria and plastids. The other two-thirds of the silver grains in electron micrographs was distributed widely, 41% over ground cytoplasm and 10% over cell walls-cell membranes. Snake venom phosphodiesterase (SVDase) extracted a cytoplasmic fraction not degraded by DNase, and did not appear to extract nuclear DNA. The SVDase-extractable fraction may be DNA or a thymidine 5'-phosphoryl group in an ester linkage with another hydroxylic compound. The nature of the nonextractable fraction is considered. Possibilities discussed are: (1) technical problems such as the binding of an acid-labile nuclear DNA in the cytoplasm; (2) non-DNA, such as breakdown products, and thymine compounds other than DNA; (3) DNA, not extractable because of the nature of its binding to other compounds or because it is a "core" resistant to DNase. Until the chemical nature of this nonextractable fraction is known, cytoplasmic label following thymidine-³H treatment cannot necessarily be considered DNA, nor the assumption made that thymidine-³H exclusively labels DNA.     

THE APPEARANCE OF ACETYLCHOLINESTERASE IN THE MYOTOME OF THE EMBRYONIC RABBIT : An Electron Microscope Cytochemical and Biochemical Study

Acetylcholinesterase (AChE) activity has been studied in the myoblast of skeletal muscle of the 9–13 day fetal rabbit. Cytochemical activity is present in the nuclear envelope and the endoplasmic reticulum, including its derivatives the subsurface reticulum and the sarcoplasmic reticulum. End product is also found in the Golgi complex of the more differentiated myoblasts. The formation of reticulum-bound acetylcholinesterase in the myoblast appears to be independent of nerve-muscle contact, since the enzyme is present before the outgrowth of the spinal nerve. The nerve lacks cytochemical end product until the myoblast is well differentiated. Possible mechanisms of spontaneous muscle contraction have been discussed. A second type of myotomal cell, which exhibits a poorly localized end product of AChE activity, has been described. The ready solubility of the enzyme or diffusibility of its end product suggests that the enzyme may be a lyoesterase. This cell may be the precursor of the morphologically undifferentiated cell which is closely apposed to the myotubes in later stages of skeletal muscle development. Biochemical studies show a significant increase in AChE activity in the dermomyotome by day 12, when many of the myoblasts are well differentiated and the second type of myotomal cell is prominent. Cytochemical studies have indicated that many of the cells in the sample lack reaction product of enzymic activity, whereas others are very active. Biochemical values, therefore, reflect the amount of enzyme in the dermomyotome as a whole, but give little information on the enzymic content of individual cells.     

An Electron Microscope Study of Cysts in the Saprolegniaceae

The structure of the zoospore cysts of various members of theSaprolegniaceae has been studied by electron microscopy. InSaprolegnia ferax, S. dioica, in Isoachlya eccentrica and I.unispora the primary cysts were smooth, whilst the secondarycysts bore stalked double-headed hooks. In S. parasitica thesecondary cysts bore tufts of longer hooks. In Protoachlya,Achlya, and Brevilegnia neither type of cyst bore hooks. InDictyuchus sterile the secondary cysts bore large spiny projections.The primary cysts in many species bore tufts of radiating hairs,and it is suggested that these are the remains of ciliated flagella.     

Electron Microscope Study of Lens Fibers

Comparative electron microscope investigations on sections of the lens cortex of the normal, mature rat, rabbit, monkey, and the normal calf reveal similar patterns of intracellular organization. The superficial lens fiber contains a nucleus, mitochondria, endoplasmic reticulum, dense granules, Golgi complex, and a quantity of small structures of low opacity which appear as filamentous and spherical configurations. Variations in number, distribution, and spatial arrangement of cytoplasmic elements in lens fibers are described. These changes in the pattern of cytoplasmic organization are concomitant with development of fibers and their displacement towards the center of the lens. Structural details of the various zones of the lens epithelium and the lens fibers are compared.     

An Electron Microscope Study of the Rat Ovum

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 µ. 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 mµ 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.     

Electron Microscope Study of the Lathyritic Rat Aorta

Six weanling rats were fed a diet containing 0.4 per cent BAPN fumarate and sacrificed after 5 to 33 days on the diet. The ascending aortae were fixed with OsO₄, embedded in methacrylate and araldite, sectioned, stained with lead hydroxide, and examined with the electron microscope. The descending thoracic aortae were examined by light microscopy. Compared with pair-fed controls, the experimentals showed definite changes which became more marked as the disease progressed. The wall became thicker with wider interlaminar spaces, radial orientation of the smooth muscle cells, progressive loss of desmosomes, and progressive increase in a dense, finely stippled material that coated the edges of the elastic laminae and extended outwards between the muscle cells and separated the ends of these cells from the laminae. This stippled material occurred at the same sites as the increase in PAS-positive and azan-positive material seen with the light microscope. There was an increase in subendothelial and interlaminar collagen, and electron microscopy clearly showed that the cells were smooth muscle and not fibroblasts. The possible bearing of the morphological changes on the formation of aortic aneurysms is discussed.     

Electron Microscope Study of the Human Neuromuscular Junction

A preliminary electron microscope study of human neuromuscular junction is presented. The biopsy material was taken from the palmarus longus, and fixed routinely in osmium tetroxide and embedded in methacrylate. The structure of the motor endings and the relationship of the synaptic vesicles to the axolemmal membrane are described. The synaptic clefts are filled with an homogeneous material in continuity with the basement membrane covering the muscle fiber. The subneural apparatus is described, and special attention is paid to a vesicular component present in the sarcoplasm of the junctional area, which differs from synaptic vesicles and is presumed to be a derivate of the sarcoplasmic reticulum.     

Electron Microscope Study of the Normal Rat Aorta

The fine structure of the normal rat aorta is described. The presence of a sub-endothelial layer, the oblique orientation of the smooth muscle cells with respect to the aortic axis, and the occurrence of desmosomes between these cells and adjacent elastic laminae, are emphasized. Lead-stained collagen presented a characteristic signet-ring appearance on cross-section. The rats examined were the pair-fed controls for the lathyritic series described in a separate communication.     

Nickel Grids Cause Astigmatism in the Transmission Electron Microscope

Nickel grids are used in various methods (e.g., in immunocytochemistry) where chemically inert grids are required. Recently (Neiss 1983) we have used formvar-coated nickel grids when removing osmium from mounted ultrathin sections with 10% periodic acid (Lewis and Knight 1977). This is possible because nickel, unlike copper, adequately withstands the oxidation necessary for osmium removal. Handling sections on nickel grids avoids the disadvantages of free-floating sections, whose use for osmium removal has been recommended by Lewis and Knight (1977, chapter 2.2.3).