ISSN:1059-910X    

DOI:10.1002/jemt.1070220202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractToday's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent‐extracted cells grown on Formvar‐coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM)Factors that are important in determining the structure and composition of detergent‐extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures.The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze‐drying (FD) is superior to critical point‐drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent‐extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze‐drayer.The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter‐coating with 1–3 nm of tungsten (W) or niobium )Nb( gives extremely fine‐grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze‐dried and then sputter‐coated within the freeze‐dryer while still froze

ISSN:1059-910X    

DOI:10.1002/jemt.1070220203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractMethods are reviewed for examination of internal cell structure by high‐resolution scanning electron microscopy and compared with the rapid‐freeze deep‐etch replica technique used in transmission electron microscopy. Rapid freezing of fresh material, followed by freezefracture, provides a theoretically attractive approach in ultrastructure studies, but the high protein and solute content of most cells prevents a deep three‐dimensional view for material frozen without some form of extraction. After discussion of other methods it is concluded that the most useful general approach, at least for cultured cells, is to first permeabilize or break open the cells in a medium which preserves the structure under study in a functional state as, for example, the movement of chromosomes along the division spindle, or transport of proteins within the Golgi region. After permeabilization, with attendant partial extraction, the preparation can be fixed, then viewed by either deep‐etch replication, or by high‐resolution scanning electron microscopy, with structure of interest revealed in deep view. © 1992 Wil

ISSN:1059-910X    

DOI:10.1002/jemt.1070220204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe application of the freeze‐fracture and cytoplasmic maceration technique in ultrastructural studies of plant cells is described. A major advantage of the technique is, that by extracting mobile cytoplasmic components from the freeze‐fractured cells, surface relief is introduced and three‐dimensional information is obtainable. The details of specimen preparation are described and the results obtained are reviewed. The use of chitosan embedding for very small or fragile specimens is described. © 1992 Wiley‐L

ISSN:1059-910X    

DOI:10.1002/jemt.1070220205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe high resolution imaging capabilities of modern field emission scanning electron microscopes require adequately improved tissue preparation procedures to prevent the collapse of macromolecular structures and the extraction of molecules. A routine cryo‐stabilization technique is described which utilizes chemical crosslinking and cryo‐dehydration for mechanical and chemical stabilization of protein and lipid structures and increase of electrical conductivity of the sample. Thiocarbohydrazide (TCH) serves as a general mordant for osmium tetroxide crosslinking. However, extensive washing after all impregnation steps is necessary to dissolve unspecific osmium black precipitations at the sample surface. Collagen I aggregates showed increased stability against collapse after TCH osmification alone, whereas pulmonary surfactant liposomes require additional freeze‐substitution in methanol and Freon 113 for stabilization during critical point drying. Environmental scanning electron microscopy (at water vapor pressures of 5–10 torr within the specimen chamber) was used to control, in the wet phase, the stabilization procedure at the level of chemical crosslinkage. It could be confirmed that tannic acid, often used to stabilize lipids, eads to artificial rearrangement of bilayered liposomes into compact presumable multilayered bodies, whereas the TCH osmification preserved liposome structures and their aggregates. The increase of electrical conductivity of sliced tissue was demonstrated on kidney. Support technologies for the cryo‐stabilization procedures are described in detail, as well as simple routines for first stabilization trials with new samples. On pulmonary tissue, the excellent preservation of alveolar shape and fine structures of intermediate forms of surfactant are described. © 1992 Wiley

ISSN:1059-910X    

DOI:10.1002/jemt.1070220206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractDetailed examination of subcellular structures in three dimensions (3D) by high resolution scanning electron microscopy (HRSEM) is now possible due to improvements in the design of the scanning electron microscope and the introduction of methods of specimen preparation using chemical removal of the cytosol and cytoskeleton by dilute osmium tetroxide. Cells which have been fixed, frozen, cleaved, thawed, and subjected to cytosol extraction display intact intracellular structures in 3D including nuclear chromatin, endoplasmic reticulum, mitochondria, and the Golgi complex at a resolution close to that of conventional biological transmission electron microscopy (TEM). Small changes in the 3D structure of subcellular components can be conveniently examined in this way in development, in a variety of physiological processes and in disease. Broad areas of the specimen can be quickly surveyed by HRSEM since sectioning is not required and specimens of comparatively large size (up to 5 mm3) can be placed in the microscope. Extraction of the cytosol with dilute osmium tetroxide (OsO4) exposes subcellular structures in relief, permitting their examination in 3D from several aspects. However, the OsO4extraction technique is limited, since significant intracellular structures, such as the cytoskeleton, vesicles, and antibody binding sites can be removed or inactivated during the cytosol removal steps. © 1992 Wiley‐Liss, I

ISSN:1059-910X    

DOI:10.1002/jemt.1070220207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe energy resolution of an energy dispersive spectrometer (EDS) equipped with an ultrathin window (UTW) and mounted at a high take‐off angle (72°) on a transmission electron microscope has been studied under a variety of operating conditions. The spectrometer resolution is close to that specified by the manufacturer, up to count rates of 400 cps. Above 400 cps the resolution deteriorates rapidly, and the MCA dead time and zero width increase. Above 10 ke V, the height of the background is much greater than expected for bremsstrahlung and shows the shape which has previously been attributed to backscattered electron flux into the detector. It is postulated that the deterioration in resolution with count rate is caused by backscattered electrons reaching the detector through the UTW. © 1992 Wiley‐Liss

ISSN:1059-910X    

DOI:10.1002/jemt.1070220208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

ISSN:1059-910X    

DOI:10.1002/jemt.1070220209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA technique, using Nuclepore polycarbonate membrane filters as a containing medium for very small volumes of ionic standard solutions, to produce homogeneous ice standards is described. The standards are suitable for use in a scanning electron microscope. The relationship between elemental X‐ray counts and ionic concentration is found to be linear. The method is rapid and simple. Minimum detectable concentrations are given. © 1992 Wiley‐Liss,

ISSN:1059-910X    

DOI:10.1002/jemt.1070220210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

ISSN:1059-910X    

DOI:10.1002/jemt.1070220211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY