高压冷冻及冷冻替代样品制备方法在体扫描电镜中的应用

高压冷冻及冷冻替代技术是一种利用高压和低温对含水组织样品进行瞬间冷冻固定和低温脱水的电镜样品制备技术,它因可以保存更加真实的细胞超微结构而常应用于透射电镜观察和分析。本文利用模式动物小鼠为材料,将这种技术应用于体扫描电镜的样品制备,并与常规化学固定的样品制备进行比较。结果显示在小鼠心肌组织中,高压冷冻及冷冻替代技术可以有效避免常规制备样品中的细胞器膜结构皱缩、细胞质分布不均匀、细胞核膜收缩形成锯齿状、甚至局部断裂等现象,使细胞超微结构更接近其生活时的状态。     

含水生物样品的环境扫描电镜观察

目前，在常规的扫描电镜中观察含水的生物样品有两种方法：（１）用戊二醛和锇酸固定细胞的骨架，再用乙醇脱水，二氧化碳临界点干燥后喷金，然后放入扫描电镜观察。（２）样品深度冷冻后使用冷冻样品台放入扫描电镜观察。以上两种制备方法都会使样品失去原来的自然面貌。...     

混合纤维素微孔滤膜用于液体藻类的冷冻扫描电镜样品制备

为了优化针对液体培养藻类细胞的冷冻扫描电镜制样条件,以铜绿微囊藻和蛋白核小球藻为材料,比较了混合纤维素微孔滤膜(混合膜)和滤纸对藻细胞的富集作用以及冷冻扫描电镜观察效果.结果表明:混合膜的孔隙较小且孔径分布均匀,正反两面均能有效吸附藻细胞.与滤纸相比,混合膜上吸附的藻细胞数量较多,细胞形态饱满,无皱缩变形,成像效果好.混合膜比滤纸更适合于微小藻类液体样品的冷冻扫描电镜样品制备.     

扫描电镜生物标本制备的超快微波处理技术

由于生物标本含有较多的水分,而扫描电镜内部是高真空的,它要求放入的标本必须干燥,但在样品空气干燥时由于液体表面张力的作用样品发生皱缩、龟裂,样品表面发生塌陷,使细胞外形发生明显改变、与生活形态的形状相差太多。为此,须对扫描电镜观察的生物样品进行固定、脱水,最后用临界点干燥器进行干燥,临界点干燥是传统的常规干燥方法。国外曾有人使用连续的微波照射固定扫描电镜样品,我们根据自己的实验经验,改进了微波固定样品的方法,使用微波照射干燥扫描电镜洋品取得很好的效果。胃和小肠,经缓冲液洗后,放入磷酸缓冲的2.5％戊二醛(pH7.3)中。实验共分三组:第1组为对照组;第2     

扫描电镜细胞样品制备方法的改进

扫描电镜已广泛用于对细胞表面结构的观察，但在细胞样品制备过程中，往往因脱水或干燥引起细胞体积或表面结构的改变，导致观察结果出现误差或失真。为了避免这类问题，我们以鱼类培养细胞为材料，对其扫描电镜样品制备技术进行了一些改进，举例如下。     

放线菌扫描电镜样品制备方法比较研究

目的:比较和筛选放线菌扫描电镜样品制备方法。方法:通过培养获得插片、菌饼和发酵液3种待处理样品,根据单因素试验设计原则采用双固定、单固定、磷酸缓冲液清洗、生理盐水清洗、蒸馏水清洗、丙酮脱水、乙醇脱水和直接表面镀金等8种方法对样品进行处理。结果:取样时间直接影响观察样品形态的丰富度;插片样品扫描获得的结果较为理想;不同的固定、清洗和脱水等处理过程也对样品的细节产生了影响。结论:插片样品直接干燥喷金扫描观察是一种简便、快速的方法,对于常规的放线菌观察与鉴定较为实用,也为其它微生物扫描电镜观察提供了参考。     

心肌纤维的ODO高分辨扫描电镜观察

本文采用ODO高分辨扫描样品制备技术,观察了小白鼠、大白鼠和犬心肌纤维表面及断面的微细结构,并讨论了高分辨扫描电镜观察法的技术特点,分析了心肌纤维的超微结构及其与功能之间的关系。技术方法按常规取材、将样品修成梭形,放入1％锇酸中固定1～2小时;继以1/15M磷酸缓冲液清洗,用25％、50％的二甲基亚砜分别浸泡20分钟,并在TF-2型冷冻割断台上将样品割断;尔后,用1％锇酸对样品作后固定,0.1％锇酸软化细胞基质,再以1～2％单宁酸对样品作导电处理,用1％锇酸对样品进行终末固定;最后,对其进行脱水、干燥、镀膜处理后,用扫描电镜观察。     

拟南芥花粉管的原位冷冻扫描电镜实验方法

花粉管在双受精过程中发挥着至关重要的作用,因此观察花粉管的生长是进行相关研究的重要手段.冷冻扫描电镜(Cryo-SEM)可实现对花粉管的原位观察,但由于花粉管样品的特殊性,常规制样方法并不适用.为此,研究者针对不同生长阶段的花粉管设计了两种冷冻扫描电镜的原位观察方法,并证实采用插秧式粘贴方法适用于柱突处的花粉管、采用液氮剥离方法适用于胎座表面处的花粉管,二种方法均可成功观察到花粉管的原位和原貌信息,为花粉管行为和相关受精机制的研究提供了有效方法.     

几种热带水果花粉的环境扫描电镜观察

环境扫描电镜可以对很多含水的、导电性差的生物样品进行直接观察，从而避免用常规扫描电镜观察时，由于生物样品经脱水、临界点干燥和导电处理而可能引起的人为变化。本文利用环境扫描电镜，对几种热带水果荔枝、龙眼、芒果、木瓜、杨桃的花粉及其相应的器官进行了直接观察，获得了较满意的结果。     

草履虫的扫描电镜观察

草履虫是原生动物门纤毛虫纲的代表动物。它对于细胞遗传和癌症的诊断有重要价值。用DMSO冷冻割断法和扫描电镜制样技术对草履虫进行处理和观察,能获得体表和体内微细结构的三维图象,提供一些新的信息。材料与方法:稻草浸液培养草履虫,2％lugol′s液缓慢地杀死,2.5％戊二醛和锇酸双固定,乙醇逐级脱水,乙酸异戊酯置换,CO_2临介点干燥,高真空浅射黄金,扫描电镜观察。断面样品用DMSO冷冻割断法。     

Histochemistry of food tissue by colour scanning electron microscopy

A low-vacuum scanning electron microscope (SEM) allows microscopy of insulating specimens without metal coating, and so preserves the intact colour information on the specimen surface. We have attempted a new approach to characterize constituent distribution of food tissues by a histochemical method utilizing a colour SEM with an optical microscope and the low-vacuum SEM through digital image processing. To observe food tissues such as brown rice and adzuki bean, a colour SEM image of the specimen that has been stained by a modified method used in optical microscope histochemistry has proved to provide information of both the microscopic structure and constituent distribution on the specimen surface.     

Scanning electron microscopy of food-poisoning bacterium Bacillus cereus using a variable-pressure SEM

A variable-pressure scanning electron microscopy (VP-SEM) with a cooling stage permits long hours of observation of water-containing specimens in their natural or close to natural state, without the conventional specimen preparations of fixation, dehydration, drying and metal coating. It reduces water vaporization and beam damage by keeping the specimens at a low temperature. We observed Bacillus cereus colonies on nutrient agar, which would shrink significantly if any conventional specimen preparation technique were used. We also studied the growing process of the bacteria on raw and steamed rice using the VP-SEM without conventional preparation techniques. Original specimens were directly mounted onto specimen holders and their backscattered electron images observed under the following conditions: specimen stage temperature, -10 degrees C; specimen chamber vacuum level, 30-70 Pa; and accelerating voltage, 15-20 kV. We recognized that the VP-SEM minimized deformation of the colonies due to shrinkage of the nutrient agar, and successfully imaged the morphology of the colonies and bacteria without a decline in bacteria number, which is apt to occur during fixation and dehydration. Also, the growth process of the bacteria on raw or steamed rice could be observed promptly, since there is no specimen preparation step.     

Influence of surrounding media on preservation of cell wall ultrastructure of Candida albicans revealed by low temperature scanning electron microscopy.

Influence of surrounding media on the surface structures of the cell wall of Candida albicans was discussed with respect to the preservation of ultrastructure during the specimen preparation for scanning electron microscopy. The fibrillar structure of the cell surface was distinctly identified by the rapid-freezing technique. It was difficult, however, to observe this structure by the conventional specimen preparation technique. The reason for the difference between these two preparation techniques was studied using a low temperature SEM. Through investigating the influence of each step of the conventional technique on the fibrillar structure, it was found that the fibrils were drastically deformed and disappeared during the dehydration step in ethanol above 80% in concentration. In order to study which physicochemical properties participated in this disappearance phenomenon, yeast cells were treated with various media: solutions in different pH ranges and at different salt concentrations, ionic solutions, surfactants, formamide, dimethyl sulfoxide, acetone and Fehling's solution. As a result, the fibrillar structure was found well preserved when the medium had an affinity for the constituent molecules of the fibrils. When without affinity, the fibrils suffered a remarkable deformation. The mechanism of this deformation is discussed in terms of molecular interaction of solute and solvent.     

准一维半导体纳米结构的电子显微学研究

本文总结了近年来我们在功能准一维纳米结构材料研究方面所获得的一些有意义的结果。借助于现代电子显微镜技术，不仅研究了硅、氮化稼、氧化锌等一维纳米材料的形貌和显微结构，还研究了其一维择优生长机理及小尺度效应。尤其是利用高能量分辨电子能量损失谱、高角环形暗场探头等先进技术，解决了一个传统X－光等结构分析手段所不能解决的难题，分析了一种SiOx/SiC复合纳米电缆的成份与结构。     

In vivo subcellular ultrastructures recognized with Hilbert differential contrast transmission electron microscopy

We describe the first application of a novel electron microscopic technique to visualize subcellular structures in a near-living state. Rapidly frozen ice-embedded cells provide the most realistic images, as they are free from artefacts induced by sample preparation methods, such as chemical fixation, dehydration, staining and sectioning. The application of the conventional transmission electron microscope to ice-embedded cell imaging, however, has been limited by the low image contrast. The recently developed Hilbert differential contrast transmission electron microscope, which exhibits an unexpectedly high contrast, akin to the differential interference contrast in visible light microscopy, enabled us to clearly discern detailed subcellular structures in ice-embedded cyanobacterial cells.     

雨生红球藻的冷冻扫描电镜制样条件初探

为了探索针对雨生红球藻的冷冻扫描电镜制样条件，以固体琼脂和液体培养的藻细胞为材料，比较了琼脂培养基直接粘台和藻液滴于预先粘台的滤纸两种粘台方式和－90℃下分别升华2 min、10 min、20 min和30 min四种升华时间对成像效果的影响。结果表明：上述两种粘台方式均可获得满意的观察效果，比较而言滤纸法效果更好；－90℃升华2 min即可将藻细胞表面完全暴露，获得满意的观察效果，而长达30 min的升华时间也不会对细胞表面结构造成损伤。     

Image quality improvement using helium gas in low voltage variable pressure scanning electron microscopy

An effective combination of the low voltage and variable pressure (VP) scanning electron microscopy (SEM) are discussed. In low voltage VP-SEM, helium gas is utilized for reducing the amount of scatter of the primary electron beam. Most samples can receive various benefits obtained from the combination of low voltage and low vacuum observation. Compared to a back-scattered electron (BSE) image in air, signal-to-noise ratio (SNR) of a BSE image taken with helium gas is 5.4 times under a pressure of 50 Pa and an accelerating voltage of 1.5 kV.     

A study of diffused bipolar transistors by electron microscopy

Shallow junction bipolar transistors have been fabricated on bulk (001) silicon starting material using a diffusion processing technology. The resultant junction depths were about 0.5 and 0.8 μ m respectively for the emitter-base and base-collector junctions. The crystallographic defects present have been detected and characterized using a combination of electrical measurements, the electron beam induced current (EBIC) mode of the scanning electron microscope (SEM) and both conventional and high voltage transmission electron microscopy (TEM).Two types of dislocations were identified in the TEM. The first consisted of an orthogonal array of sessile, diffusion-induced edge dislocations located about halfway down in the heavily doped emitter. The second type consisted of 60° glissile dislocations that looped down from the orthogonal array and in some cases penetrated the emitter-base junction.Observations of a number of 60° dislocations yielded a good correlation between their depth and their contrast when imaged in the EBIC mode of the SEM. The 60° dislocations locally retard the emitter diffusion when they lie near to the emitter-base junction. Local breakdown effects under reverse bias were observed at the sites of these dislocations.