Biological specimen preparation for transmission electron microscopy

Biological Specimen Preparation for Transmission Electron Microscopy (1998). A.M. Glauert, P.R. Lewis. In: A.M. Glauert (Ed). Practical Methods in Electron Microscopy, Vol 17. London: Portland Press, 326 pp. £39.50 paperback; ISBN 1 85578 060 7     

高水分、富含淀粉植物组织的扫描电镜制备技术优化

应用常规高真空扫描电子显微镜观察生物样品必须经过脱水和干燥处理,但无论采用临界点干燥还是冷冻干燥方法,都存在样品表面不同程度失真的问题。植物高水分、富含淀粉组织样品经处理后,容易出现淀粉流失、细胞壁变形等现象,从而造成扫描图像粗糙,无法获得真实的细胞内部结构。本文通过对CO_2临界点干燥、化学固定样品冷冻干燥和新鲜样品冷冻干燥3种扫描电镜样品制备技术中后期制样进行机械断裂和液氮脆断改进,优化出两种植物高水分、富含淀粉组织的扫描电镜样品制备方法:(1)样品首先进行FAA化学固定,经冷冻干燥后用液氮脆断,对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞结构完整,细胞壁整齐,淀粉粒和蛋白轮廓明确,可用于分析淀粉粒和蛋白颗粒在细胞内的分布。(2)新鲜样品直接进行冷冻干燥,经液氮脆断后对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞壁整齐,淀粉粒轮廓更清晰,并且无蛋白颗粒干扰,用于分析淀粉粒在细胞内的分布更加理想。     

Considerations of specimen damage for the transmission electron microscope, conventional versus scanning

We have previously shown that the transmission scanning electron microscope is capable of giving a resolving power equal to that of a conventional electron microscope, that it can be used to provide all the same contrast modes, but that it offers the advantage of new forms of contrast and can provide direct numerical outputs (Crewe &; Wall, 1970a,b; Crewe et al. 1970; Crewe, 1970).One question that we have not previously discussed is that of specimen damage, but in view of the similarity in performance between the two types of machine it has become important to do so.Recent remarks of Scherzer (1971) have been widely misinterpreted as indicating that the scanning microscope causes more specimen damage. However, he has confined his attention to the highest conceivable resolution of 0·4 Å, where we agree with his general conclusion (barring advances in scanning microscope technology). As we will demonstrate, the conclusion is not valid for normally attainable resolutions.     

Rapid preparation of uncoated biological specimens for scanning electron microscopy.

We have developed a relatively rapid glutaraldehyde-tannic acid (GTA) and osmium tetroxide (OsO4) fixation procedure which permits many types of uncoated biological specimens to be examined in the scanning electron microscope (SEM) at 20 kV without the occurrence of charging. Most specimens taken one day can be examined in the SEM the following afternoon. Types of specimens successfully treated were perfused adult and embryonic rat tissues, confluent human skin fibroblast tissue cultures, plant roots, flowers, seeds, some garden insects, and microcolonies of salivary streptococci. Cells in suspension and extracted human teeth did become electron conductive when treated with the GTA procedure. Most suspended cells must be centrifuged between each solution and the GTA procedure increases the preparation time for these cells. Extracted teeth are usually simply dried and coated. Therefore, the usual SEM preparation techniques are shorter and perhaps more useful for these types of specimens.     

Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

A simplified method for the preparation of rotifers for transmission and scanning electron microscopy

Tor TEM and SEM prepatations, rotifers are placed in little panels made of plastic Beem capsules normally used for embedding, with their conical parts cut off and closed by plankton filter cloth. Thus, the risk of losing animals is considerably reduced.     

Microwave processing for scanning electron microscopy

The normal processing of biological samples for Scanning Electron Microscopy, includes treatment with aldehyde (1 to 2 hours), postfixation with Osmium (1 hour), followed by dehydration in a ascending grade of ethanol (30 a 100%), 10 to 15 minutes in each step, and finally drying. This procedure takes at least 8 hours. In this work, samples of mosquitoes (Aedes), protozoa (Tritrichomonas muris), bacteria (Clostridium oceanicum), murine liver, and small intestine were processed in the same manner in a domestic microwave oven for two minutes at 20% of its maximum power. The complete procedure from the initial fixation to dehydration in 100% ethanol was reduced to one hour with good preservation of the ultrastructural details of the specimens.     

A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy

A new rapid procedure for preparing soft internal tissues from insects that allows air drying was found to compare favorably with tissues prepared by critical point drying. In the new procedure, tissues were fixed in 1% glutaraldehyde, dehydrated through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes, and air dried. Tissues prepared by both the HMDS treatment and by critical point drying were coated with gold for scanning electron microscopy. Tissues prepared by the HMDS treatment did not shrink or distort upon air drying and excellent surface detail was preserved. The HMDS treatment required about 5 minutes, whereas the critical point drying procedure required about 1.5 hours.     

Freeze-drying from tertiary butanol in the preparation of endocardium for scanning electron microscopy.

The scanning electron microscope appearances and shrinkage of blocks of canine endocardium prepared by freeze-drying directly, by freeze-drying after replacing tissue water with tertiary butanol (2-methyl propan-2-ol) and by critical point drying were compared. All three methods demonstrated endothelial cells which showed nuclear prominences, microvilli and intercellular boundaries. The microvilli varied in size and number from dog to dog but were generally less well defined in specimens freeze-dried from water. Shrinkage due to t-butanol dehydration was significantly less than that which occurred in ethanol in the critical point drying method. Overall the reduction in surface area was significantly less in specimens freeze-dried directly at -65 C (6.8%) than in those dried from t-butanol at -20 C (15.4%) and those prepared bly critical point drying (22.1%). However the amount of shrinkage observed in t-butanol treated tissue was not significantly different from that which was critical point dried. It was not possible to distinguish between comparable samples prepared by these two methods on the basis of their scanning electron microscopic appearances. Thus the relative simplicity and convenience of the t-butanol method, together with its saving of time, its use of standard freeze-drying equipment and the avoidance of ice-crystal artefact justify its consideration as an alternative method of preparing wet biological tissue for scanning electron microscopy.