SECRETORY CELLS OF LILY PISTILS. I. FINE STRUCTURE AND FUNCTION

The fine structure of the cells which line the canal of Lilium longiflorum pistils confirms the secretory function which has been ascribed to them. The cells differ in structure from the secretory cells which cover the stigma surface and are therefore referred to as canal cells rather than stigmatoid cells. Their most striking feature is an elaborate wall, 8–14 μ in maximum depth, on the side facing the canal, which with associated structures, we term the secretion zone. Pollination, which triggers chemotropic activity in the style and secretory activity in the canal cells, is not correlated with marked changes in the fine structure of the canal cells. The canal cells appear to fit well into that category which Gunning and Pate have termed “transfer cells.”     

ELECTRON MICROSCOPE STUDIES OF THE MICROVILLI OF HELA CELLS

Microvilli of HeLa cells cultured in vitro were preserved for electron microscopic examination at different stages of routine cultivation procedures. By a double-embedding technique, vertical sectioning for electron microscopy was possible. It revealed that, although the microvilli were present on all sides of the cell in the dispersed stage and in the attached stage, they were not present on the bottom of the cell when it was stretched on the surface of the dish. When the cells were grown in dense colonies, they were found on top of each other, and microvilli were present on all sides, except on the bottom surface of those cells in contact with the dish. We achieved a more dramatic demonstration of the microvilli by developing a surface-replica technique which retains their spatial arrangement and permits characterization of the distribution of their number, length, and diameter.     

ELECTRON MICROSCOPE OBSERVATIONS ON FROZEN-DRIED CELLS

To explore the problem of artefacts which may be produced during usual fixation, dehydration, and embedding, the authors have examined pancreas, liver, and bone marrow frozen at about -180°C., dried, at -55 to -60°C., embedded in methacrylate, sectioned, and floated on a formol-alcohol mixture. By these treatments the labile structure of living cells can be fixed promptly and embedded in methacrylate avoiding possible artefacts caused by direct exposure to chemical fixatives. Cell structures are ultimately exposed to a fixative when the sections are floated on formol-alcohol, but at this stage artefacts due to chemical fixation are expected to be minimized, as the fixatives act on structures tightly packed in methacrylate polymer. In the central zone of tissue blocks so treated, the cells are severely damaged by ice crystallization but at the periphery of the blocks the cell structure is well preserved. In such peripherally located cells, elements of the endoplasmic reticulum (ER), Palade's granules, homogeneously dense mitochondria, and nuclear envelopes and pores, can be demonstrated without poststaining with OsO₄. The structural organization in the nucleus is distorted by vacuolization. The mitochondrial membranes and cristae, cellular membrane, and the Golgi apparatus, however, are detected only with difficulty. The Golgi region generally appears as a light zone, in which some ambiguous structures are encountered. After staining the sections with OsO₄ or Giemsa solution, an inner mitochondrial structure which resembles the cristae seen in conventional OsO₄-fixed specimens appears, but the limiting membrane is absent. Treatment with OsO₄ or Giemsa solution also renders more distinct the membrane of the ER and Palade's granules but not the Golgi apparatus and cellular membrane. Treatment with ribonuclease results in the disappearance of Palade's granules. On the strength of these observations the authors conclude that OsO₄ fixation gives a satisfactory preservation of such cell structures as the nuclear envelope, endoplasmic reticulum, and Palade's granules, though it may induce slight swelling of these cell components.     

ELECTRON MICROSCOPE AUTORADIOGRAPHY OF ERYTHROID CELLS USING RADIOACTIVE IRON

The "circle analysis" method of Williams (1969) and a new improved method employing hypothetical grains described in the previous paper have been used to analyze the distribution of autoradiographic grains over erythroid bone marrow cells labeled with radioactive iron, ⁵⁵Fe. The resolution obtainable with this isotope was determined by measuring the distribution of grains about a thin line source. This distribution was also used in calculation of the circle size for the Williams's analysis and the distances of hypothetical grains for the new method. The new method provides estimates for the amount of activity in the regions of condensed and extended nuclear chromatin and for the concentration of isotope at the junction between these two areas. The possible significance of activity in this junctional region is discussed.     

百合离体生殖细胞骨架的扫描电镜观察

从百合的花粉管分离出来的生殖细胞,经表面活化剂Ｔｒｉｔｏｎ Ｘ－１００ 及核糖核酸酶、硫酸铵离析处理。离析后的细胞经临界点干燥,扫描电镜观察显示：在离体生殖细胞的胞质内有一个非常复杂的支架网络系统。这一网络系统有内外两层：外层（靠近细胞膜）网络结构紧密,纤维束粗长;内层（靠近核）网络结构疏松,纤维束短细。一些间接证据显示,这一支架网络系统可能为微管骨架     

AN ELECTRON MICROSCOPE STUDY OF THE GLAND CELLS OF THE MINK ENDOMETRIUM

Portions of mink endometrium in delayed implantation, early postimplantation, and pseudo pregnancy were fixed in buffered osmium tetroxide with sucrose, or potassium permanganate. After rapid dehydration the portions of endometrium were embedded in either methacrylate or epoxy resin. Examination of the cells from the body of the glands of the endometrium of delayed implantation revealed the presence of prominent terminal bars, numerous secretion granules, and membrane discs in the apical region of the cell. In the supranuclear and infranuclear regions, mildly dilated cisternae of the endoplasmic reticulum were present, and in many cells unusually large mitochondria were seen. Numerous changes were noted in the gland cells of the post implantation stage. The endoplasmic reticulum in the basal region was extensively dilated, and the nuclei were situated more centrally. Giant mitochondria were no longer present. The large secretion granules were not present, but smaller granules were seen, especially in the Golgi region. Some of the Golgi cisternae were dilated and the pattern of parallel membranes was consequently less distinct. It is suggested that gland cells in the postimplantation and pseudopregnancy stages exhibit evidence of greater secretory activity than those in the delayed implantation stage.     

AN ELECTRON MICROSCOPE STUDY OF SILVER NITRATE REDUCTION IN LEAF CELLS

Brown , W. V., H. Mollenhauer , and C. Johnson . (U. Texas, Austin.) An electron microscope study of silver nitrate reduction in leaf cells. Amer. Jour. Bot. 49(1): 57–63. Illus. 1962.—As reported earlier in many studies, AgNO₃ is reduced quickly by the living chloroplasts of angiosperms. Electron microscope study has resolved the conflict of opinions concerning the exact location of the silver particles. Reduction of AgNO₃, as indicated by location of silver particles, occurs within the chloroplasts but not within the grana or pure stroma; it appears to be associated with the intergranal (also called stroma) lamellae. Silver particles are formed also at the surfaces of the cell wall, both in the middle lamella and at the inner surface, and also within plasmodesmata. It is concluded that chlorophyll is probably not involved directly in the reduction. There is some slight support for the popular hypothesis that ascorbic acid may be the chief reducing agent.     

ELECTRON MICROSCOPE REPLICAS FROM THE SURFACE OF A FRACTURE THROUGH FROZEN CELLS

A technique is described, which is new in certain details, for fracturing frozen cells and taking replicas from the fracture surface. This technique has been developed as a cytological preparative technique complementary to those currently used in electron microscopy. The present paper describes results for human red cells, chosen for preliminary work to facilitate recognition of artifacts. The cells are suspended in 20 per cent glycerol, 0.9 per cent NaCl solution, allowed time to equilibrate, and rapidly frozen in a small chamber plunged into propane at -196°C. The chamber is kept at -196°C., and fractured in vacuo, in such a way that the fracture plane runs through the frozen suspension, and through the cells in its path. After fracture, the chamber is brought to -105°C., still in vacuo, to etch the fracture surface, and a platinum-carbon replica is deposited on the surface from a carbon arc. The replica is subsequently cleaned in concentrated alkaline solution and examined in the electron microscope. The outlines of the fractured cells can be recognised. There are indications that in areas of the replica which correspond to the interior of a cell, individual haemoglobin molecules can be seen, and in favourable cases the arrangement of some of the four molecular subunits.     

ELECTRON MICROSCOPE RADIOAUTOGRAPHIC IDENTIFICATION OF SEROTONIN-SYNTHESIZING CELLS IN THE MOUSE GASTRIC MUCOSA

This study correlates the fine structure of mouse gastric endocrine cells with their ability to synthesize serotonin (5-HT) from 5-hydroxytryptophan (5-HTP). Mice were sacrificed 2 hr after the intravenous injection of 5-HTP-³H or 5-HT-³H. Their stomachs were processed for light- and electron microscope radioautography in a manner which retained labeled 5-HT while washing out other labeled substances. Stomachs from additional mice were incubated in vitro with 5-HT-³H and processed similarly. All morphologic types of mouse gastric endocrine cells exhibited a similar facility to incorporate exogenous 5-HTP and to convert it to 5-HT which was bound intracellularly. Differences in densities of silver grains observed over endocrine cells suggested that individual endocrine cells indeed varied in their ability to synthesize and/or to bind 5-HT; such variations, however, were not reflected by differences in fine structure, with the exception that endocrine cells with few granules always contained little newly synthesized 5-HT. The newly synthesized 5-HT was associated with the intracellular granules. The gastric endocrine cells were not labeled by exogenous 5-HT-³H, whereas mast cells were labeled by either 5-HT-³H or 5-HTP-³H administration. The findings of the present study support the position that the gastric endocrine cells represent a single cell type, at least in respect to serotonin metabolism—that the argyrophil or argentaffin reactivity of these cells merely reflects their amine content at a given time.     

四君子汤抗骨骼肌疲劳作用的定量细胞化学和电镜观察

本文采用细胞化学技术及单细胞定量对运动疲劳小鼠股四头肌不同类型肌纤维的成分进行分析,实验结果显示：经服四君子汤恢复后的三种肌纤维类型的糖原含量、琥珀酸脱氢酶（ＳＤＨ）、乳酸脱氢酶（ＬＤＨ）的活性明显高于疲劳组,超微结构的改变则位于线粒体的变化,提示四君子汤对解除骨骼肌的高度疲劳、增强体质具有非常重要的作用。     

IDENTIFICATION OF MAST CELLS IN THE SCANNING ELECTRON MICROSCOPE BY MEANS OF X-RAY SPECTROMETRY

In mixed populations of rat peritoneal fluid cells, the mast cells can be differentiated from other cell types in the scanning electron microscope by virtue of the X-ray emission referable to their sulfur content. Both stationary probe and X-ray mapping are feasible; the amount of sulfur eliciting a signal is estimated to be about 8 x 10^-18 g.     

THYMIDINE-3H ELECTRON MICROSCOPE RADIOAUTOGRAPHY OF OSTEOGENIC CELLS IN THE FETAL RAT

The proximal tibial epiphyses of 18–21-day-old fetal rats have been studied by thymidine-³H radioautography. The results reveal that the label is incorporated into two types of osteogenic cells: (a) a spindle cell type (A cells) with characteristics generally associated with matrix production, including an extensive development of the endoplasmic reticulum and the presence of large intracellular accumulations of a dense, finely granular material, morphologically identifiable as glycogen; and (b) a rounded cell type (B cells) with morphological features similar in degree and kind to those of the developing neutrophilic leukocyte, including an abundance of free ribosomes and mitochondria and a complex Golgi apparatus associated with dense specific granules, morphologically identifiable with primary lysosomes. These results, along with the occurrence of recognizable, labeled, immature, perivascular forms of both of these A and B type cells, lead to the conclusion that the specialization of osteogenic cells into osteoclasts and osteocytes may involve separate pathways of cytodifferentiation.     

豚鼠脊髓前角神经元线状溶酶体的电镜酶细胞化学研究

本研究采用电镜金属盐法──三偏磷酸酶（ＴＭＰａｓｅ）细胞化学方法探讨了豚鼠脊髓前角神经无线状溶酶体（ＮＬＹ）的存在及其酶细胞化学特点。ＴＭＰａｓｅ是港酶体的标志酶之一。本研究首次以偏磷酸钠代替三偏磷酸钠为ＴＭＰａｓｅ的底物,很好地显示了ＮＬＹ的结构。ＴＭＰａｓｅ反应产物虽高电子密度的黑色铅盐沉淀,不仅分布于圆形溶酶体,也可见于ＮＬＹ,同时,在高尔基复合体的部分板层也有酶活性分布,可能酶是经高尔基复合体加工后输送至溶酶体。ＮＬＹ在神经元胞体,突起及突触中均有分布,并且从胞体到神经终末均有ＮＬＹ和线粒体相贴现象,提示酶是由依赖于线粒体提供运动能量的ＮＬＹ从胞体输送到神经终末,这可能和ＮＬＹ参与神经递质的降解及神经元代谢物质的处理密切相关。     

RESOLUTION IN ELECTRON MICROSCOPE RADIOAUTOGRAPHY : II. Carbon14

Experimental resolution values, half distances (HD), were determined for electron microscope radioautography with ¹⁴C as the source of radioactivity. These were about a factor of 1.5–2 times higher than for tritium. Grain distributions normalized in units of HD were found to fit the "universal" curves previously obtained for tritium.