ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03082.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYThe theoretical and experimental evidence in favour of cryofixation and freeze‐substitution are critically reviewed. The solubility of macromolecules in water is due to the hydration shells. Their behaviour at different temperatures and the consequences of their removal during the processing for embedding are explained. Gelation prior to the transfer into solvents prevents macromolecules aggregating. During substitution at low temperatures, DNA is gelled, justifying the use of the term cryofixation. It is proposed that the preservation of hydration shells at the lowest temperature, and their transformation into minute gaps after a rise of temperature, facilitates the exhibition of epitope

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03083.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYWe have developed cryofixation and ultra‐low‐temperature molecular distillation drying as a method for preparing biological samples for electron microscopic analysis. To validate this approach, we have investigated the relationship between the drying characteristics and ice phases present within frozen samples.Two sample types were investigated. In the first, pure deuterium oxide (D2O), or heavy water, was vapour condensed under vacuum conditions onto a gold‐coated copper sample holder held at −175 or −110°C. Additionally, D2O was slow‐rate cooled from room temperature under an ultra‐pure dry nitrogen gas atmosphere. The second sample type was rat liver biopsies from animals after 5 days of feeding with D2O loaded water and ultra‐rapid cooling by metal‐mirror cryofixation. Ice forms present in the latter samples, determined by electron diffraction of frozen‐hydrated cryosections, were amorphous, cubic, and hexagonal.Drying of samples was achieved using a molecular distillation configuration with continuous, microprocessor‐controlled sample heating. The vacuum contents of the drying column were monitored by residual gas analysis (RGA) throughout the drying cycle.D2O vapour in the vacuum chamber, as analysed by RGA, was found to increase in a phasic manner across a broad temperature range. These phases had characteristic onset temperatures and could be removed sequentially. For condensed D2O samples, these onset temperatures were — 160, — 148, — 125 and — 90°C. Rat liver samples also demonstrated phasic drying patterns which were more complex than those detected with pure D2O samples.Ultrastructural analysis of samples cryofixed and dried in this manner demonstrated a morphology consistent with the ice phases demonstrated in the frozen‐hydrated cryosections. This, together with the RGA results, suggests the absence of devitrification or ice crystal g

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03084.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYFreeze‐substitution of biological material in pure acetone followed by low‐temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry‐cut sections shows that diffusible cellular thallium ions (Tl+) of Tl+‐loaded muscle are localized at similar protein sites in freeze‐substituted as in frozen‐hydrated preparations. A comparison of X‐ray micro‐analytical data obtained from freeze‐dried cryosections and sections of freeze‐substituted normal (potassium‐containing) muscle shows that K+ion retention in the freeze‐substituted sample is highly dependent on the freeze‐substitution procedure used; so far, in the best case, about 67% of the cellular K+is retained after freeze‐substitution in pure acetone and low‐temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze‐substitutio

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03085.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYPreparing cellular structures for visualization by high‐resolution scanning electron microscopy (SEM) is a multi‐step process which includes fixation, dehydration, drying and metal coating. Drying and metal coating are limiting for high‐resolution work. Commonly, the dried samples are exposed to the air before they are inserted into a metal coating apparatus, thereby exposing them to moisture and the accompanying risk of rehydration, which may cause changes in the supramolecular structure. We have modified a freeze‐dryer to accommodate a magnetron sputtering head, in order to sputter‐coat the frozen‐dried samples while still in the drying chamber in the cold, a process we call cryosputtering. A layer of 1·5 nm of tungsten was cryosputtered onto whole mounts of cytoskeletons from detergent‐extracted human glioma cells or fibroblasts and the specimens were examined by high‐resolution SEM and transmission electron microscopy (TEM). To reduce the effects of backstreaming oil from the vacuum system, a turbomolecular pump backed by a two‐stage rotary vane pump was connected to the drying‐coating chamber. This pump system provides a high vacuum, making it possible to dry the specimens at — 90°C/183 K, thus reducing the risk for recrystallization of water. Furthermore, the high vacuum minimizes the negative effects of contaminants, which can be deposited onto the specimen surface and affect the quality of the metal coat f

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03086.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYElectron microscopy of biological macromolecules embedded in vitrified ice films suffers from serious problems caused by excessive inelastic scattering, beam‐induced movements of the specimen, deformation of the molecules by adsorption at the water‐air interface and insufficient mechanical stability of the films. We have built an environmental chamber to control temperature and humidity independently in order to produce ultrathin water films (<20 nm) spanning holes with diameters of 20 μm to 1 mm. The surface tension of the water films was reduced by adding lipid monolayers, thus prolonging the usable time for thinning of the film and avoiding adsorption artefacts in the embedded material. After cryofixation in ethane a carbon film was evaporated on each side of the specimen to stabilize the ultrathin ice—lipid film. Mechanical stability and charging effects could successfully be reduced by this preparation method. Collapsing water films could be cryofixed and the shape of the hole was analysed. By the eccentricity of the elliptical holes an estimation could be made of the burst velocity of the rim of the hole and of the cooling rate of the cryofixation p

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03087.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYWe investigated the possibility of vitrifying temperature‐sensitive lipid phases as well as (small) biological specimens. From a suspension of unilamellar vesicles, prepared from dipalmitoyl‐phosphatidylcholine (DPPC), thin aqueous films were formed at various temperatures. With cryo‐electron microscopy vesicles were found to be smooth, rippled and faceted or faceted only, depending on the temperature of thin‐film formation (318, 312 and 296 K respectively). The morphology and the electron diffraction patterns indicate that membranes can by physically fixed by vitrification in their high‐temperature configuration and studied at low temperature by cryo‐electron microscopy. This finding suggests that it may also be possible to preserve, in their original state, the more complex membrane systems found in living organisms by initiating rapid‐cooling at a physiological temperature. This was explored by vitrification of thin films formed on specimen grids with (human) blood platelets adhering to collagen fibres. Low‐temperature observation with an acceleration voltage of 120 kV revealed subcellular details. More details were observed when using higher accelerating voltages (200 and 300 kV) of the electron beam. The results presented in this paper illustrate the great potential of cryo‐electron microscopy in the study of membrane dynamics, both in relatively simple model membrane systems and in more complex biological

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03088.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYThe surface layer of the archaebacteriumSulfolobus acidocaldariushas been re‐investigated exploring various image analysis and image processing strategies. Straightforward correlation averaging correcting for translational disorder only, and lattice unbending in conjunction with filtration in the Fourier domain gave equivalent results. Careful examination of cross‐correlation functions as well as multivariate statistical analysis of individual unit cells revealed the existence of interpenetrating domains with intrinsic p3 symmetry but rotated with respect to each other by 60° and separated by twin boundaries. Non‐discriminative averaging over twinned crystal patches generates a higher (p6) class of symmetry emphasizing the importance of selective ave

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03089.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYN‐Paraffin was used as a test specimen for evaluating the relative merits of 400‐kV versus 100‐kV electron microscopy in recording data for electron crystallographic analysis of beam‐sensitive materials. The parameter used for comparison, the relative contrastR, is the ratio of amplitudes from the computed Fourier transform of images and amplitudes from an electron diffraction pattern from the same crystal.Rwill thus be a measure of the contrast from an experimental image relative to that of a perfect image. Electron diffraction patterns and bright‐field images were recorded at 400 kV at a specimen temperature of −167°C. Using the flood‐beam imaging technique the bestR‐value is 0 08 for all reflections in the resolution zone from 4 to 3 Å. This value is equivalent to that found at 100 kV. In the resolution zone from 3 to 2 Å we have foundR —0 02. Using the spot‐scan imaging technique, on the other hand,Rwas measured to be 0·42 for the reflections between 4‐ and 3‐Å resolution. This amount of relative contrast is 1·7 times that observed at 100 kV. Reflections at 3–2 Å displayed anR‐value of 0 05. Besides obtaining higherR‐values when applying the spot‐scan imaging technique at 400 kV, we observe a higher yield of images with isotropic diffraction and/or higher resolution reflections. Various contrast‐attenuating factors, including the modulation transfer function of the photographic film and the cryo‐holder, envelope functions for spatial and temporal coherence and lens and high‐tension instabilities, the contrast transfer function and lastly the radiation damage effects, have been considered in interpreting the observed image contrast. Overall, use of 400 kV in combination with spot‐scan does offer important improvements in contrast levels, which can be very useful in determining the thr

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03090.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYThe rational design of cryopreservation protocols for living tissues demands an understanding of the mechanisms of mass transport between cells and their environment throughout the entire process. We have developed a new microscope stage to enable a specimen to be viewed continuously during a preservation protocol, including the addition and removal of cryoprotective additives and freezing and thawing. The specimen is contained in a sealed chamber having inlet and outlet ports for admitting and collecting perfusate solution, the entire volume of which may be exchanged with a time constant of 1–5 s, depending on the solution viscosity. The temperature of the active area of the stage is regulated by the standard techniques of convection cryo‐microscopy over a range in excess of 50 to — 100°C.A series of experiments has been performed on this system to measure the osmotic behaviour of rat pancreas islets during the addition and removal of dimethyl sulphoxide at temperatures between 25 and — 10°C. The technique involves mounting a single islet onto the low‐temperature stage so that it is constrained from lateral movement by a specially sized mesh. Both the system temperature and chemical composition are monitored and controlled simultaneously and independently; as a consequence, virtually any defined cryopreservation protocol may be imposed on the specimen. For making permeability measurements, the bathing medium of the specimen may be changed very rapidly to produce a defined osmotic stress. Alternatively, the specimen may be subcooled to a specific and fixed subzero temperature, at which point ice is nucleated in the extracellular medium, creating a near instantaneous change in composition. The temporal alteration in specimen size is monitored by video microscopy and quantified by computer vision analysis methods. One of several mass transfer models is fitted to the data to estimate the membrane permeability based on the assumption of either transport dominated by the movement of water or simultaneous coupled flows of water and cryoprote

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03091.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY