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.     

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。     

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.     

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.     

Simplified preparation of mycoplasmas, an acholeplasma, and a spiroplasma for scanning electron microscopy.

A simple, effective procedure was developed for scanning electron microscopic examination of mycoplasmas and similar organisms. Cultivation of several mycoplasmal species, an acholeplasma, and a spiroplasma in broth media in Leighton tubes with cover slips resulted in attachment of the organisms to the cover slips. The attached cells were easily processed for either scanning electron microscopy or light microscopy. By eliminating the need for centrifugation, which was used in previously described techniques, physical stress on the cell is minimized. The effects of different preparative procedures on the morphology of Mycoplasma gallisepticum are described.     

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.     

Cryo-preservation for scanning electron microscopy avoids artefacts induced by conventional methods of specimen preparation

Cryo-preservation of tissues for scanning electron microscopy avoids artefacts associated with critical point drying and freeze-drying (solvent extraction and cell distortion). Motile specimens are arrested immediately, cells remain fully hydrated, delicate structures are undisturbed and conductive coating with metals or carbon is achieved at low temperatures.     

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.     

Fixation of platelets for scanning and transmission electron microscopy.

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

Device to facilitate freeze-drying specimens for scanning electron microscopy.

Thin specimens such as tissue culture monolayers were freeze-dried in a vacuum evaporator without thawing by using a 13 mm thick aluminum disk cooled with liquid nitrogen just prior to processing. This disk, 95 mm in diameter, easily accommodated 18 specimens suitable for mounting on 12.5 mm specimen mounts. The specimens were gold coated immediately after drying without breaking the vacuum thereby saving the time usually needed for a second evacuation. Vero cells processed in this way displayed their delicate surface structures.