不同包埋剂对半薄切片甲苯胺蓝染色效果的影响

目的:探讨不同包埋剂对半薄切片甲苯胺蓝染色效果的影响。方法:切取大鼠的坐骨神经及肾脏组织,经戊二醛-锇酸双重固定,乙醇、丙酮梯度脱水后,分别用Epon812和Eponate12包埋聚合,利用超薄切片机制备厚度为1.5μm的半薄切片,切片经甲苯胺蓝染色,在光镜下观察半薄切片的染色效果。结果:Eponate12包埋的半薄切片经甲苯胺蓝染色后,颜色更鲜艳,微细结构更清晰,图像反差更好。结论:Eponate12包埋的半薄切片经甲苯胺蓝染色后更有利于在光镜下观察组织的微细结构。     

热暴露大鼠脑组织超微结构改变

机体在热环境中的耐受能力有一定限度,超过限度就会发生中暑,中暑常常伴有谵妄和昏迷等神经系统症状,因此热暴露对脑组织的影响特别受到注意。光镜观察已经积累了许多材料,但是电镜观察尚未见有报导,为此选择大鼠脑组织神经细胞和毛细血管为对象观察超微结构改变。大鼠在干球温度38—40℃、湿球温度31—32℃的高温仓中受热致死,取5只作电镜观察,平均受热时间215分钟,平均最高体温41.7℃。死亡大鼠立即开颅取大脑与小脑皮质部份组织,以戊二醛固定,再以锇酸固定,Epon812包埋,半薄切片作Giemsa染色,超薄切片作铅铀染色。另有3只大鼠不受热,在室温条件作对照观察,活杀取材,处理相同。     

免疫金法与ABC法在电镜包埋前染色之比较

包埋后免疫电镜术虽能精确定位抗原,但难度大。本文用胶体金标记法与ABC法对人鼻中线恶网瘤组织在树脂包埋前作抗人T细胞免疫染色,并对两种方法进行比较。简述如下:临床活检鼻中线恶网组织,经4％多聚甲醛加1％戊二醛4℃固定,振动切片(50μm),PBS冲洗,在预先硅化的载玻片上作免疫染色:鼠抗人T细胞McAb(Dako公司出品)孵育过夜;金标羊抗鼠抗体(10nm,军科院基础所出品)室温下孵育1小时,再将切片倾入小瓶,经1％锇酸后固定,常规脱水包埋及超薄切片,不作铀铅复     

甲状腺胶性腺瘤及乳头状癌的超微结构

采用电镜观察肿瘤细胞,提高细胞结构的分辨能力是肿瘤形态学研究的重要手段。本研究对比观察了甲状腺胶性腺瘤及乳头状癌的超微结构,并探讨了不同超微结构特征对癌的诊断意义。材料来自北医第一附属医院外科手术室的手术切除标本,共12例胶性腺瘤及5例乳头状癌。组织切成1mm小块固定在3％戊二醛及1％锇酸后固定,环氧树脂618包埋超薄切片经醋酸铀及枸椽酸铅双染色。JEM100CXⅡ电子显微镜观察,剩余标本固定在10％福尔马林液中作常规病理组织切片及检查。讨论:甲状腺胶性腺瘤和乳头状癌的超微结构有明显不同,尤其显著的是核的变化。胶性腺瘤有较大,而且     

胃印戒细胞癌浸润转移与免疫超微结构特征的关系

胃印戒细胞癌是一种较其它类型胃癌恶性程度更高的肿瘤 ,其 5年生存率较低主要与肿瘤的生物学特征有直接关系。本文通过免疫组化及电镜技术对 5 3例胃印戒细胞癌进行功能与超微结构相结合的观察 ,对印戒细胞癌浸润性的特征进行探讨。材料与方法　收集 1 995年至 1 999年 2月病理标本中的 5 3例胃印戒细胞癌组织 ,标本均经 1 0 %中性福尔马林固定 ,石蜡切片 ,HE染色、免疫组化癌胚抗原 ( CEA)及层粘连蛋白 ( LN) S- P法检测。同时取其中 6例留取电镜样品 ,戊二醛、锇酸双重固定 ,低粘度包埋剂浸透包埋 ,半薄切片定位 ,超薄切片铀 -铅双重…     

电镜包埋块快速制备中污染物的实验观察

临床诊断电镜工作的开展 ,必然要求改变以往周期长的制样过程 ,而包埋块制备过程所花时间最多 ,人们用微波聚合、高温聚合等条件以缩短聚合时间 ,并在实际工作中得以应用 [1]。但常为了快速 ,而在样品固定到包埋前的处理中忽视了某些细节 ,造成包埋块制备中的人为污染 ,影响电镜观察 ,这些污染无法通过返工来弥补。本文就此污染物产生进行模拟实验与探讨。材料与方法正常小鼠乙醚麻醉后 ,快速取肾组织入 3%戊二醛 - 1 .5 %多聚甲醛的前固定液中 ,后固定液用 1 %锇酸 - 1 .5 %亚铁氰化钾 ( 1∶ 1 ) ,70 %酒精饱和铀液块染过夜 ,系列酒精、丙…     

大熊猫精子的超微结构

近年来,人们研究大熊猫的生态、生理、饲养和繁殖,取得了一些成绩。1978年大熊猫人工授精繁殖在北京动物园试验成功,开辟了新的繁殖途径。目前国外有人研究大熊猫精子的发生及发育状况,搜集冷冻精液活力等数据,但对精子超微结构的研究较少。笔者对大熊猫精子进行电镜观察,了解精子的超微结构特征,为提高大熊猫繁殖率提供资料。方法将新鲜的大熊猫精液离心,取沉淀用琼脂预包埋,经戊二醛与四氧化锇双重固定,按常规方法包埋、制作超薄切片,铀铅双重染色,JEM—100cX电镜观察。结果和讨论电镜下大熊猫精子主要由头部、颈部和尾部组成。头部有细胞核和顶体,细胞核电子密度较高,     

扁桃酸酰胺作用阴道滴虫的电镜观察

扁桃酸酰胺(Mandelic Amide)已证实为桃叶膏抗滴虫的有效成份,为探讨此成份的杀虫机制,作者曾作了光镜下的形态观察,而对电镜下的超微结构改变未见有过记载。本文就电镜下的观察结果报导如下。观察方法是在培养48小时的滴虫培养管内加入40mg/5ml扁桃酸酰胺,培养48小时后制备样品。另外取一滴虫培养管不加药品作对照组,并与实验管同时进行样品制备。样品制备是将上述两种标本,先以2.5％戌二醛液在室温下作一级固定,15分钟后,用pH7.4磷酸缓冲液水洗1小时,再入1％四氧化锇二级固定2小时。用乙醇进行系列脱水,用环氧树酯包埋处理48小时,作超薄切片,切片用醋酸铀和柠檬铅作双染色,随后在H—600型电镜下观察拍片。     

哮喘豚鼠支气管超微结构变化特征研究

为了更好地了解哮喘超微形态结构特征 ,我们对豚鼠哮喘模型的超微结构作一些电镜观察研究 ,对其超微结构特征作一些阐述。材料和方法取 1 0只体重 30 0～ 5 0 0 g的健康豚鼠 ,雌雄个半 ,随机分组 ,分成哮喘组和正常组 ,每组 5只。按徐叔云方法[1] 致豚鼠哮喘 ,哮喘诱发成功后 2 4～ 48小时 ,用 2 .5 %戊二醛活体插管灌注豚鼠肺部 ,最后完全杀死两组豚鼠 ,取出肺 ,在左右叶肺中 ,取不同部位 ,直径约为 0 .5～ 2 .0 mm的支气管 ,2 .5 %戊二醛和 1 %锇酸双固定 ,常规电镜包埋、切片 ,H- 6 0 0 A型透射电镜观察。结果和讨论电镜观察正常组豚鼠…     

电子显微镜检查在肌肉疾病诊断之应用

从1981年11月起,作者等在神经科搜集肌肉疾病之肌肉切片样本合计684例,切片样本主要做组织病理,组织化学诊断,或必要时做电子显微镜检查。切片方法通常为经皮切开,或针刺切片。切片之肌肉组织,分为三部分,一者在以液态氮冷至-160℃之isopentane内急速冷冻,以便做组织冷冻切片,病理染色及组织化学染色;另一部分组织培养或生化分析;第三部分则电镜下做超微细构造之观察。电镜检查用之肌肉组织切成1立方毫米小块固定于3％glutaraldehyde内,而后再以锇酸二度固定染色,脱水后渐次包埋於epon内,切1nm之厚片,选择将镜检之部位,切100nm之薄片,并以uranyl acetate及lead citrate双重染色,而后用日立500型电子显微镜观察。     

Staining of intestinal goblet cells with ruthenium red in semithin sections.

For a correlative light and electron microscopy of intestinal goblet cells, postembedding staining with ruthenium red (RR) was performed in epoxy-embedded sections. Tissue blocks were fixed in buffered aldehyde and embedded in a mixture of Quetol 651, nonenyl succinic anhydride (NSA), methyl nadic anhydride (MNA), and DMP-30. Sections at 0.4-0.5 micron in thickness were mounted on grids and were treated with an aqueous solution of RR followed by osmium tetroxide, uranyl acetate and lead citrate. Postembedding staining of epoxy sections revealed the interaction between RR and anionic groups by both light and electron microscopy. Light and electron microscopic observation of identical sites in semithin sections was successful for the correlations of colored reaction with electron density.     

A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy

Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.     

果蝇的透射电镜制样方法研究

果蝇作为一种模式生物，被广泛应用于遗传学、发育的基因调控、人类疾病机制等研究。透射电镜能够观察细胞的超微结构及其变化，是研究果蝇细胞生物学的重要方法之一。本实验研究发现，果蝇样本固定前用双面刀片快速切下头部，固定液能快速有效地固定其体内组织成分，防止内部组织溶解；使用琼脂预包埋果蝇样本，将其修整为长方体，增加其自重，固定其包埋方向，为后续目标位置的超薄切片起到定位作用。     

Correlative light and electron microscopic observations on ectopic neurons in the cerebellum of dreher mutant mouse

The structure of ectopic neurons in the cerebellum of dreher mutant mouse was investigated by correlative light and electron microscopic observations. Tissue blocks were fixed in buffered aldehyde and embedded in a mixture of 2-hydroxypropyl methacrylate, Quetol 523, and methyl methacrylate. Sections at 0.4-0.5 microns in thickness were examined by electron microscopy after observation under a light microscope. By comparing the electron images with those of light microscopy in the same sites, the structures of ectopic cells were confirmed. Ectopic Purkinje cells were arranged with cell bodies that contained an oval, spherical or wrinkled nucleus without deep invagination and the thin layers of endoplasmic reticulum at the perinuclear regions. Granule cells were ectopically matured in the external granular layer and within the cluster at the cortical region. This method provides a useful procedure for understanding structures of the cerebellar neurons of the mutant.     

Morphological and histochemical analyses of living mouse livers by new 'cryobiopsy' technique

A new 'cryobiopsy' (CB) technique has been invented for freezing the functioning livers of living mice in vivo without stopping their blood circulation. Livers of anesthetized mice were pinched off with pre-cooled CB forceps and immediately plunged into isopentane-propane cryogen. They were routinely freeze-substituted in acetone containing paraformaldehyde for light microscopy (LM) or osmium tetroxide for scanning electron microscopy (SEM). By freeze-fracturing some of them with a scalpel in liquid nitrogen before the freeze-substitution, well-preserved tissue areas were exposed only for SEM. They were either embedded in paraffin wax for LM or infiltrated with t-butyl alcohol followed by freeze-drying for SEM. Serial paraffin sections were stained with hematoxylin-eosin (HE) or histochemical periodic acid-Schiff (PAS) reaction. By HE-staining, the tissue surface areas were often compressed with the CB forceps and sinusoidal erythrocytes became aggregated side by side. In slightly deeper tissue areas, however, hepatic sinusoids were widely open with flowing erythrocytes. Lots of PAS-reaction products were well preserved in hepatocytes of the CB specimens. On the contrary, they were unevenly distributed in hepatocytes of conventionally quick-frozen specimens, and often lost in those of the conventionally dehydrated specimens. By SEM, some cell organelles, such as mitochondria and endoplasmic reticulum, and also dilated fenestrae of endothelial cells, open Disse's spaces and bile canaliculi appeared to be under normal blood circulation in the prepared CB samples. The new CB technique would be easy and useful for repeated examination of functioning organs of a living animal.     

Ultrastructure of Staphylococcus aureus as revealed by microwave fixation.

The ultrastructure of the cell wall of Staphylococcus aureus was examined at electron microscopic level using new chemical fixation techniques during microwave irradiation and the results obtained were compared with those obtained by other conventional techniques. By using microwave fixation the concentric circular or zipper-like structure was observed in the cell wall. This structure was observed also with the spray-freeze-etch technique but not in thin section of the cells chemically fixed by conventional technique. For chemical fixative, glutaraldehyde is more advantageous than OsO4 as a concomitant fixative during microwave irradiation and postfixation by OsO4 is unnecessary and rather harmful for the preservation of the ultrastructure. The function of the observed structure is briefly discussed.     

Three-dimensional reconstruction by scanning electron microscopy from serial epoxy resin semi-thin sections after ion-etching

Three-dimensional reconstruction was performed using scanning electron micrographs of serial semi-thin sections of Epon embedded specimens. Connective tissue in a rabbit ear chamber was fixed in glutaraldehyde and osmium tetroxide, and then embedded in Epon. One-microm-thick serial sections were cut with a diamond knife, mounted on glass slides and stained with toluidine blue. After observation with a light microscope, the sections were ion-etched with an ion-spatter coater. Following double staining with uranyl acetate and lead citrate, the consecutive sections were ion-coated with platinum. Each serial section was photographed with a scanning electron microscope. Profiles of a blood vessel and fibroblasts were digitized with a computer and computer reconstruction of the blood vessel was performed. Three-dimensional reconstructions showed that the newly formed blood vessel was a cylinder-like, bare endothelial tube with a rather smooth outer surface. Fibroblasts were situated around the endothelial tube. Several openings were found in the endothelial tube, suggesting the morphological feature of high permeability and fragility in newly formed blood vessels. The availability of three-dimensional reconstruction from scanning electron micrographs of serial semi-thin epoxy resin sections was discussed; structures of interest can be reconstructed (1) quickly and easily, (2) without skilful techniques, and (3) almost at the level of ultrastructure.     

煤岩样品TEM超薄切片制备方法研究

本文作者以泥炭、褐煤、烟煤及无烟煤为例,探讨了超薄切片制备方法对不同煤阶样品的适用性。在采用包埋切片法制备煤岩样品TEM超薄切片时,首先通过浸解离析方法和HCl、HF逐级化学浸蚀方法使煤中自然共生组合的有机显微组分离析及脱除无机矿物质;利用光学显微镜(透射/反射模式)镜检离析显微组分后,使用SPI812树脂对挑出的单一显微组分块体渗透及包埋聚合。显微组分块体形态学及嵌布矿物成分分析使用扫描电子显微镜的低真空二次电子像模式和EDS面分布分析模式;利用透射电子显微镜检查超薄切片效果。实验表明采用上述方法制备煤岩TEM超薄切片样品的成功率较高,并且能够比较真实的再现显微组分的微观结构。     

基于块容量标签的密文图像可逆数据隐藏方法

密文图像的可逆数据隐藏技术既能保证载体内容不被泄露,又能传递附加信息。本文提出了一种基于块容量标签(block capacity label, BCL)的高容量密文图像可逆数据隐藏算法。该方案在图像加密之前进行预处理,首先将图像分为两个区域：参考像素区域和预测像素区域。然后将预测像素区域分为不重叠的块,根据所提出的算法确定分块的BCL,在对图像进行加密之后嵌入BCL,生成加密图像;在秘密数据嵌入阶段,根据BCL和数据隐藏密钥嵌入秘密数据。实验测试了BOWS-2数据集,平均嵌入容量为3.806 8 bpp,与现有方法相比,该方法可以获得更高的秘密数据嵌入容量,并可以实现原始图像的完美重建。     

基于嵌入矩阵的二值图像隐藏方法

描述了一种基于嵌入矩阵的二值图像信息隐藏方法,该方法将图像交叉分块后,计算各子块的可修改分值,根据可修改分值的大小选取子块的中心像素作为数据嵌入的位置;数据嵌入时,使用基于嵌入矩阵的信息隐藏方法,在嵌入同等数据量的同时,可有效地减少对原图像数据的修改.