摘要:

SUMMARYThe preparation and high resolution observation of frozen hydrated thin sections has been studied by transmission electron microscopy (TEM and STEM) on model systems, including pure water, protein solutions, catalase crystals, myelin sheath and various tissues. The state of the ice is determined by electron diffraction. Mass measurement in the electron microscope is used to determine section thickness and control hydration. An adequate depth of vitrified material for sectioning can be obtained from many biological suspensions or untreated tissues. Frozen hydrated sections around 100 nm thick can be produced under optimal conditions from vitreous ice or from vitrified biological samples. Sectioning, transfer and observation in the electron microscope is feasible without alteration of the sample hydration or its initial vitrification. Biological structures can be preserved and observed down to 10 nm. Under favourable working conditions, specimen compression during sectioning and electron beam damage are the factors limiting high resolution observations.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04225.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04224.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYIt is important for future ultrastructural preservation studies to freeze biological specimens as rapidly as possible. Finite element numerical techniques have been used to compare different cooling block materials used in the quick freeze ‘slamming’ method. It is concluded that a pure silver block at an optimum initial temperature of about 15.6 K will produce the quickest cooling and will give rates approximately 50% higher than if the block were initially cooled to 4.2 K.A copper block will produce the fastest cooling rates when the block is initially at liquid nitrogen temperature of 77.3 K provided the section being studied is taken no more than about 30 μm from the specimen‐block interface.Composite metal blocks can further reduce the total specimen cooling time compared to a mono‐block at the same initial temperature but the cooling rate near the interface will be slower.Future methods for specifying cooling rates within thin biological samples should be standardized and must be based on a combination of finite element numerical analysis and the measured temperature at the rear surface of the sample. Thermal contact resistance between cooling block and specimen and between specimen and the rear face temperature sensor must be

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04226.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYThe anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer pH 7.4. Some of the blocks are post‐fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic‐embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive techni

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04227.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYA comparative study of various cryofracturing techniques has been conducted on the mammallian myocardial cell. Quench freezing of fresh or fixed tissue in melting Freon 22 resulted in severe cellular damage due to ice crystallization. Fixation with Karnovsky's fixative prior to quenching had no modifying effect on the size and distribution of the ice crystals. The crystals were orientated primarily in the direction of the long axis of the myofibrils, manifested as empty tube‐like structures in the scanning electron microscope (SEM). Regular cross‐bridging often seen at the Z‐band levels indicated that ice crystals, at least in some portions of the cells, were confined within the sarcomere. Within the same cell the size of the ice crystals could vary considerably.Treatment of the tissue with polyvinylpyrrolidone (PVP) prior to rapid freezing had no noticeable cryoprotective effect. The surface of the thin layer of PVP surrounding the freeze dried tissue appeared amorphous in the SEM. However, the first evidence of ice crystallization was found a few micrometres under the surface.The freezing artefacts were completely circumvented if the cryofracturing was carried out on ethanol‐impregnated or on critical point dried material. While the first method resulted in a smooth fracture plane passing through the cell structures, the intracellular fracture plane of the critical point dried material followed the surface of the cell organelles.Separation of the cell organelles caused by freezing or by critical point drying revealed thread‐like structures extending from the mitochondrial surface. Re‐examination of SEM‐processed material in the transmission electron microscope (TEM) revealed that these structures were part of the sarcoplasmic reticulum (SR), and that a close contact between the SR and the outer mitochondrial membrane existed. TEM of conventional prepared material revealed that strands of electron‐dense material, here named ‘mito‐reticular junctional fibres’, bridged the narrow gap between the mitochondrial surface and the SR. It is suggested that these fibres have a specif

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04228.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYThe relative advantages and disadvantages of high resolution and other more general imaging techniques are considered and the potential of a multipurpose, moderate resolution, TEM with EELS is assessed.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04229.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYA set of techniques for preparing transmission electron microscope specimens from sections of thin (≤ 1 mm overall diam.) multifilamentary wires (filaments being approximately 5 μm diam.) are described. Results illustrating the successful uniform thinning of the several phases present in ‘bronze‐route’ Nb3Sn a

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04230.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYThe topology of faceted void‐like defects (‘voidites’) in gem‐quality type la diamonds has been revealed by high resolution phase‐contrast electron microscopy. Interpretation of the images in terms of a Fresnel contrast mechanism allows the defects to be identified as octahedral in shape. Image contrast is sensitive to defocus and other parameters, and Fresnel fringe interference effects may generate ‘superlattice’

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04231.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYThe resolution of modern transmission electron microscopes reaches the physical limits imposed by lens aberrations and energy width. One of the many conditions to be fulfilled, the alignment of illuminating and imaging beam onto the coma‐free objective axis, is particularly discussed here since axial coma cannot be detected by the usual resolution‐checking methods. Space consumption of specimen stages prevents the full utilization of the magnetic saturation limit only in the 100 keV range. With higher energies, this handicap is obviated, and some additional advantages can be gained which promote material investigations at atomic resolution, and which are presently utilized in instrumental research projects. High resolution with biological specimens has up to now been unsuccessful because of radiation damage. Optimum utilization of all electrons scattered at the specimen must thus be given priority over optical resolution. Important instrumental requirements are minimum exposure beam control, imaging modes with high collection efficiency, and recording devices with high detection quantum efficiency connected on‐line to image processors. A remarkable decrease in beam sensitivity of organic crystals, by more than one order, has been found by cooling the specimen down to 4 K which, by the use of superconducting lenses, can be combined with both ultra high vacuum and the stability requirements for high resolution. Yet up to now, such protection has not been achieved with He cryostates in conventional lenses, perhaps because a temperature increase even of only a few degree K is harmful.Purely magnetic imaging energy filters are about to be developed to a high optical quality but have been employed so far in only a few high resolution instruments. Such filters allow removal of the inelastic background and thus improvement of contrast of images of low‐Z specimens, particularly in the dark field mode.Finally, some ‘non‐conventional’ projects have made progress. Correction of spherical and chromatic aberration by multipole lenses offers a chance to improve remarkably the resolution in the 100 keV range, to extend the bandwidth of phase contrast transfer and to obtain highly resolved information about inelastic images when an energy filter is also applied. Electron holography provides possibly useful large area phase contrast, particularly if the electron energy is decreased, which may be of great benefit in investigations of unstai

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04232.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY

摘要:

SUMMARYThis paper describes the morphometric changes liver samples undergo in the course of fixation, dehydration, infiltration and embedding in different mixtures of water‐soluble plastics. The plastics used were: three different mixtures of glycolmethacrylate (GMA) and the commercially available material JB4.Buffered formaldehyde fixation did not produce significant morphometric changes in the liver specimens. Dehydration obviously affects the volume of the liver specimen (linear shrinkage about 9.3%). The dehydration is followed by an infiltration phase. During this phase a slight swelling (linear, 2–5%) occurs. The final polymerization of the plastic resulted in a further linear shrinkage of 1–2%.The influence of different technical factors on the stretching of sections of pure plastic and of plastic embedded liver appeared to be considerable. The difference in stretching between 2 and 3 μm sections has been studied. A significant influence of temperature upon section stretching was noted. Sections of all plastic mixtures stretched at a temperature of 293 K showed 3% more linear stretch than at a temperature of 333 K. Differences between the four plastic mixtures are discussed.Correction factors must be used in morphometrical and stereological investigations (see for review: Weibel, 1979). It was concluded that in the application of water‐soluble plastics as embedding media it is essential to apply a standardized procedure, particularly in the cutting and stretchi

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1983.tb04233.x

出版商:Blackwell Publishing Ltd    

年代:1983

数据来源: WILEY