Ultrastructural transformation of gastric parietal cells reverting from the active to the resting state of acid secretion revealed in isolated rat gastric mucosa model processed by high-pressure freezing

To elucidate a functional transformation of gastric parietal cells, we have newly developed an isolated rat gastric mucosa model whose parietal cells exhibited a reverting process from the active to the resting state of acid secretion. Briefly, the parietal cells were treated with cimetidine following prior stimulation of acid secretion in the model, and cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, immunohistochemistry of H(+)/K(+)-ATPase demonstrated a progressive translocation of H(+)/K(+)-ATPase from the apical to the cytoplasmic region. The ultrastructure of parietal cells at 5 min in the reverting phase was quite similar to that of maximally stimulated one. However, the apical microvilli of intracellular canaliculi (IC) changed bulbous by degrees, resulted in complete occlusion of IC at 60 min in the reverting phase. The apical membranes were subsequently internalized into the cytoplasm forming unique penta-laminar membranes. Interestingly, at 90 min in the reverting phase, the penta-laminar membranes formed a number of multilamellar autophagosomes that were intensely labeled for H(+)/K(+)-ATPase. Then, the parietal cells exhibited well-developed Golgi apparatus and lysosomal compartments involving the multilamellar membranes at 105 min, and mostly reverted to their resting conformation at 120 min in the reverting phase. Corresponding to the ultrastructural changes of microvilli, the immunohistochemistry of ezrin showed a dissociation of ezrin from the apical region at 30 min in the reverting phase. The present findings provide new insights into the functional transformation in gastric parietal cells reverting to their resting conformation.     

Electron microscopy of pathogenic yeasts Cryptococcus neoformans and Exophiala dermatitidis by high-pressure freezing

A high-pressure freezing method was used to observe the ultrastructure of pathogenic yeasts, Cryptococcus neoformans and Exophiala dermatitidis, after freeze-substitution and ultrathin sectioning. The method well preserved the cell structure in its natural state, since the capsule, cell wall, plasma membrane, nucleus, outer and inner nuclear membranes, nuclear pores, nucleolus, mitochondria, mitochondrial membrane and cristae, vacuoles, endoplasmic reticulum, Golgi apparatus, spindle pole body, ribosomes, lipid droplets, microtubules, actin filaments, and glycogen granules were clearly visible. The method was shown to freeze cells as deep as 0.1 mm by sectioning the sample perpendicular to specimen surface. The quality of the cell image was similar to that obtained by a rapid freezing method when compared using the same materials. Thus, high-pressure freezing would be useful for making serial ultrathin sections for three-dimensional analysis of cells, which should give basic information of structure and function of pathogenic yeast cells necessary for finding an effective therapy for mycoses.     

Effects of different fixation and freeze substitution methods on the ultrastructural preservation of ZYMV-infected Cucurbita pepo (L.) leaves

Different fixation protocols [chemical fixation, plunge and high pressure freezing (HPF)] were used to study the effects of Zucchini yellow mosaic virus (ZYMV) disease on the ultrastructure of adult leaves of Styrian oil pumpkin plants (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) with the transmission electron microscope. Additionally, different media were tested for freeze substitution (FS) to evaluate differences in the ultrastructural preservation of cryofixed plant leaf cells. FS was either performed in (i) 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate, (ii) 0.01% safranin in anhydrous acetone, (iii) 0.5% glutaraldehyde in anhydrous acetone or (iv) anhydrous acetone. No ultrastructural differences were found in well-preserved cells of plunge and high pressure frozen samples. Cryofixed cells showed a finer granulated cytosol and smoother membranes, than what was found in chemically fixed samples. HPF led in comparison to plunge frozen plant material to an excellent preservation of vascular bundle cells. The use of FS-media such as anhydrous acetone, 0.01% safranin and 0.5% glutaraldehyde led to low membrane contrast and did not preserve the inner fine structures of mitochondria. Additionally, the use of 0.5% glutaraldehyde caused the cytosol to be fuzzy and partly loosened. ZYMV-induced ultrastructural alterations like cylindrical inclusions and dilated ER-cisternae did not differ between chemically fixed and cryofixed cells and were found within the cytosol of infected leaf cells and within sieve tube elements. The results demonstrate specific structural differences depending on the FS-medium used, which has to be considered for investigations of selected cell structures.     

Cytochemistry of gastric parietal cells with high-pressure freezing followed by freeze-substitution

Gastric parietal cells were examined for changes in their ultrastructure and distribution of the proton pump during feeding and fasting states in rats. The fundic glands from rats fed ad libitum or fasted with free access to water were cryofixed using high-pressure freezing followed by freeze-substitution in acetone containing osmium or acrolein and then embedded in Epon 812 or Lowicryl K4M resin, respectively. Excellent ultrastructural preservation was achieved. During the feeding state, intracellular canaliculi and numerous microvilli were well developed, while tubulovesicles were poorly developed. In contrast, during the fasting state, the microvilli in the narrowed space of the intracellular canaliculi were tightly packed and the tubulovesicles were enlarged. Ultrathin sections were immunostained with antibodies against the alpha- and beta-subunits of the proton pump, H+ x K(+)-ATPase, using the immunogold method. The labelling was strong and clearly localized in comparison with that obtained using the conventional chemical-fixation method. Each subunit was localized on the membrane of the microvilli, intracellular canaliculi and tubulovesicles. The distribution of subunit proteins varied between the two states. During ad libitum feeding, the immunolabelling was localized strongly on the membranes of the microvilli and intracellular canaliculi. In contrast, the labelling was strong on the tubulovesicle membrane in the fasting state. The results obtained with each anti-subunit antibody by H+ x K(+)-ATPase immunostaining revealed differences in distribution and labelling density between the feeding and fasting states.     

应用高压冷冻-冷冻置换技术研究受Potyvirus侵染的寄主细胞超微结构

应用高压冷冻-冷冻置换技术（HPF-FS）制备了马铃薯Y病毒属的小西葫芦黄花叶病毒（ZYMV）和大豆花叶病毒（SMV）侵染寄主植物叶细胞的超薄切片样品,并与传统化学固定方法进行比较。透射电镜观察结果显示：病毒粒子和内含体的形态分布在两种方法处理的样品中无明显差异,但高压冷冻样品的细胞超微结构精细,细胞壁与细胞质之间边界清晰,质膜平滑而完整,细胞基质丰富;细胞核和叶绿体、线粒体等形态饱满,高尔基体潴泡结构及分泌小泡清晰,在ZYMV感染细胞的叶绿体中观察到囊泡结构。而化学处理的样品普遍存在细胞结构的收缩和变形,特别是质膜褶皱,与细胞壁分离,叶绿体呈梭形,片层结构发生改变,高尔基体潴泡结构及分泌小泡相当少见。实验结果有利于正确区别病毒所引起的细胞病理变化和化学处理而产生的人工假象。     

骨骼肌兴奋收缩偶联时肌膜下小泡的超微结构变化

目的：从毫秒级功能变化水平实时观察骨骼肌肌膜下小泡在收缩潜伏期内的时相-形态变化。方法：采用双红外线探测器-计算机控制的电刺激-超低温快速冷冻固定同步技术,对电刺激后的蟾蜍骨骼肌组织作快速冷冻固定,冷冻置换,微波浸透包埋和超薄切片,在透射电镜下观察该骨骼肌细胞在电刺激后0．0ms,4．6ms,24ms的超微结构变化。结果：未加刺激的骨骼肌细胞的肌膜下仅见少量小泡分布;施加刺激4．6ms后肌膜下出现大量小泡,并由3～8个小泡融合成聚合体;24ms后小泡急剧减少,仅残留少量小泡紧靠肌膜下。结论：骨骼肌兴奋．收缩偶联发生时,肌膜下出现大量小泡。     

骨骼肌兴奋收缩偶联时肌浆网膜Ca2+通道的研究

目的：从毫秒级功能变化水平实时观察骨骼肌肌浆网、T-管在收缩潜伏期内超微结构的时相—形态变化。方法：采用双红外线探测器—计算机控制的电刺激—超低温快速冷冻固定同步技术,对电刺激后的蟾蜍骨骼肌组织作快速冷冻固定,采用透射电镜对骨骼肌在电刺激后0．8ms,2ms,4．6ms,10．8ms和18．4ms的超微结构变化进行观察。结果：刺激0．8ms后,肌浆网和T-管未见明显改变。刺激2ms后,SR内出现电子密度较大的物质。刺激4．6ms后,肌浆网膜内外侧可见一一对应的电子密度大的物质。刺激10．8ms后,SR与T-管形态又恢复原状,SR内电子密度大的物质消失。结论：骨骼肌兴奋-收缩偶联发生时,肌浆网的形态发生改变,肌浆网内出现电子密度较大的物质且逐渐向靠近T-管的方向移动。     

骨骼肌兴奋收缩偶联时肌浆网内Ca2+释放的形态-功能变化研究

采用双向红外线探测器－计算机控制的电刺激－超低温快速冷冻固定同步技术,透射电子显微镜,X－射线能量色散谱及Ca^2 细胞化学技术,从超微结构形态学角度,实时研究并获取骨骼肌兴历－收缩偶联时,肌浆网内Ca^2 释放及肌浆网的形态改变。研究表明：1．骨骼肌组织在收缩潜伏期内,肌浆网膜与T－管膜接触。2．在潜伏期（＜10ms)内,肌浆网内的钙离浓度随时间经历的延长而减少。3．骨骼肌组织在收缩潜伏期开始0．8ms时,在T－管外周围（纳米处）,有Ca2 分 ,丰,可以认为骨骼肌兴奋收缩偶联时,T－管外存在Ca^2 结合位点。     

高压冷冻及冷冻替代技术在不同生物样品超微结构中的应用

高压冷冻仪在固定样品时,高压和冷冻基本是同步发生的,使得生物样品在数毫秒的时间内被瞬时固定,保证其内部结构接近于生理状态,有效减少了化学固定法中的化学试剂对细胞结构的破坏,极大程度保存了细胞在近似自然状态下的超微结构.本文分别采用化学固定法(CF)和高压冷冻固定法(HPF)对小鼠的睾丸组织、Hela细胞和酵母进行了样品...     

电刺激—冷冻固定法研究兴奋收缩偶联时骨骼肌的超微结构形态变化

兴奋－收缩偶联时，肌浆网中的钙离子是通过何种途径被刺激而释放的？偶联发生时期形态变化与其功能变化存在着什么的关系？这是细胞生物学、生理学几十年来最感兴趣的研究热点之一。     

Clinical applications of the quick-freezing and deep-etching method to human diabetic nephropathy

Mesangial expansion and glomerular basement membrane (GBM) thickening were not different between normoalbuminuric (NA) and microalbuminuric (MA) type 2 diabetic patients. The quick-freezing and deep-etching (QF-DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. In the present study, the QF-DE method was applied to the renal biopsy from 6 type 2 diabetic patients without definable renal diseases other than diabetic nephropathy. Four patients were NA and the other two were MA. Three control specimens were normal parts in surgically resected kidneys of renal cell carcinoma. Replica membranes were prepared by the QF-DE method as previously described. By the QF-DE method, both GBM middle layer and mesangial matrix (MM) were composed of polygonal meshwork structures. The mesh pores of GBM and MM were more enlarged in size and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The diameters of mesh pores in the diabetic patients were significantly larger than those in the control subjects. In conclusion, the QF-DE method could be applied to needle renal biopsy and the present study has firstly clarified the difference of ultrastructural changes between NA and MA type 2 diabetic patients, which had not been disclosed by the conventional electron microscopy, were revealed by the QF-DE method.     

郎格罕氏组织细胞增生症的电镜研究

目的：探讨郎格罕氏组织细胞增生症（LCH）的超微病理特点,提供Birbeck颗粒来源的线索。方法：结合光镜、免疫组化及临床资料。对2例LCH进行了超微结构观察。结果：朗格罕组织细胞胞浆内可见棒状、网球拍状Birbeck颗粒,这些细胞免疫组化CDla阳性。结论：LCH的诊断须见到典型的郎格罕氏细胞（Langerhans cell,LC）LC细胞,电镜下找到Birbeck颗粒,或CDla（010）阳性。Birbeck颗粒可与细胞膜相连续,细胞膜凹陷可在胞质内形成Birbeck颗粒。     

The origins and evolution of freeze-etch electron microscopy

The introduction of the Balzers freeze-fracture machine by Moor in 1961 had a much greater impact on the advancement of electron microscopy than he could have imagined. Devised originally to circumvent the dangers of classical thin-section techniques, as well as to provide unique en face views of cell membranes, freeze-fracturing proved to be crucial for developing modern concepts of how biological membranes are organized and proved that membranes are bilayers of lipids within which proteins float and self-assemble. Later, when freeze-fracturing was combined with methods for freezing cells that avoided the fixation and cryoprotection steps that Moor still had to use to prepare the samples for his original invention, it became a means for capturing membrane dynamics on the millisecond time-scale, thus allowing a deeper understanding of the functions of biological membranes in living cells as well as their static ultrastructure. Finally, the realization that unfixed, non-cryoprotected samples could be deeply vacuum-etched or even freeze-dried after freeze-fracturing opened up a whole new way to image all the other molecular components of cells besides their membranes and also provided a powerful means to image the interactions of all the cytoplasmic components with the various membranes of the cell. The purpose of this review is to outline the history of these technical developments, to describe how they are being used in electron microscopy today and to suggest how they can be improved in order to further their utility for biological electron microscopy in the future.     

Ultrastructural characteristics of chondroblastomas

The ultrastructure of chondroblastomas and two new ultrastructural peculiarities typical of the given form of tumours are described. Being typical but not constant these peculiarities may be an additional argument indicating that such cells may be regarded as chondroblastoma cells. The ultrastructure of these cells is similar to that of normal epiphyseal chondrocytes and differs from the ultrastructure of chondrosarcoma cells.     

Detection of glycoconjugates by lectin gold labelling, silver enhancement, and scanning electron microscopy

A method for detecting glycoconjugates on cell surfaces in scanning electron microscopy is described. Terminal saccharides were specifically recognized by a lectin conjugated to biotin, and, after incubation with an anti-biotin antibody conjugated to colloidal gold, silver enhancement was used to produce deposits large enough to be detected in standard scanning electron microscopes. Secondary electron images revealed the ultrastructure of the tissue investigated, while backscattered electron images showed the distribution of lectin binding sites. Using digital recording and processing, the two channels were combined in colour-encoded images. The new method brings together lectin histochemistry and scanning electron microscopy and thus allows the three-dimensional distribution of glycoconjugates to be analysed at an ultrastructural level.     

露蜂房蛋白对急性髓细胞白血病患者骨髓单个核细胞超微结构的影响

目的：观察中药露蜂房蛋白(NVP)成份对急性髓细胞白血病(AML)患者骨髓单个核细胞(BMMNC)超微结构的影响。方法：采用体外细胞培养,加入不同浓度露蜂房蛋白成分作用于AML患者BMMNC,培养72h收集细胞,常规制备超薄切片,透射电子显微镜下观察AML患者BMMNC的超微结构。结果：露蜂房蛋白各处理组细胞核染色质浓缩、边集,呈新月形或环状或细胞核碎裂呈块状。线粒体出现空泡样变及髓样变。结论：中药露蜂房蛋白成份有明显诱导AML患者体外培养BMMNC凋亡的作用,并呈现典型的细胞凋亡超微结构改变。     

Ultrastructural organization of the blood lymphoid cells in hairy cell leukemia

The cytochemical, immunological, and ultrastructural peculiarities of the hairy-cell leukemia cells are studied. The hairy cells can be identified by characteristic of ultrastructure of organellae: numerous thin processes on the plasma membrane, large cytoplasmic area, well developed granular endoplasmic reticulum, numerous vesicules and electron dense granules, a well differentiated lobulated nucleus with a large nucleolus in which the granular component predominates.     

Culture prior to transplantation preserves the ultrastructural integrity of monkey pancreatic islets

Transplantation of pancreatic islets represents a promising way of curing type I diabetes (insulin-dependent diabetes mellitus). Culture enables the survival of endocrine tissue awaiting islet transplantation and reduces islet immunogenicity prior to xenografting. In this study, attempts were made to preserve the monkey islets in culture for 7 days and to study the ultrastructure by electron microscopy. The islets were isolated from monkey pancreas by the collagenase digestion method and were separated from acinar cells by dextran density gradient centrifugation. These islets were preserved in a humidified atmosphere of 5% carbon dioxide and 95% air for 7 days. The culture medium used was CMRL-1066. After 7 days of culture the islets were processed for light and electron microscopic studies, which revealed that the cultured islets were intact and maintained their structural integrity. Semi-thin sections of the cultured islets showed morphology with occasional structural alterations at the periphery. Dithizone staining of the cultured islets showed crimson red colour, proving that the islets were pure and without any exocrine contamination. Electron microscopy showed that the cultured islets had well-preserved alpha-, beta- and delta-cells. Different cell types of the monkey pancreatic islets were identified by the presence of their characteristic secretory granules. The ultrastructural characteristics present in hormone-synthesizing cells, i.e. rough-endoplasmic reticulum, Golgi apparatus, mitochondria and secretory granules, were observed as in native islets.     

自噬小体膜来源的形态学分析

目的:从形态学角度探寻自噬小体形成过程中的膜来源及自噬小体的超微结构特征.方法:应用透射电镜技术整理分析本中心收到的用于自噬研究的细胞标本,对自噬发生不同阶段及疑似自噬小体结构的图片进行分析.结果:在细胞内发现众多自噬小体结构,内质网、线粒体、细胞核和高尔基体均可形成类似自噬小体结构.结论:细胞内多种膜结构可能都是自噬小体的膜来源.