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PubMed1987

X-ray microanalysis of growth cartilage after rapid freezing, low temperature freeze drying and embedding in resin.

This paper reviews the work undertaken on the X-ray microanalysis of growth cartilage particularly in relation to matrix calcification. Attention is focused on the methodology available to accurately localize and retain the elements of interest. A method is described which involves rapid freezing, low temperature freeze drying in a custom built device, and embedding in Spurr resin. The results show that little tissue damage occurs and that elements of physiological interest are accurately localized at the sub-cellular level. Where damage occurs to chondrocytes as a result of freezing, however, dense intra mitochondrial granules are seen suggesting a phase transformation takes place resulting in the precipitation of calcium phosphate. Further improvements may be made in retaining the more labile elements such as K by using resins which polymerize at low temperature.

Animals↗

PubMedDate not supplied

Freeze-etching and freeze-fracture structural features of cell envelopes in mycobacteria and leprosy derived corynebacteria.

The structural properties of the cell wall and cell membrane of several mycobacteria and of Leprosy Derived Corynebacteria are investigated by freeze-etching and freeze-fracture. In all cases the freeze-fracture split the cell wall in two asymmetric halves. The cell wall fracture faces of the mycobacteria are characterized by a filamentous network which vary with respect to the amount and complexity among microorganism of the same species and even more of different species. In LDC the structure organization of the cell wall and cell membrane differs from that of mycobacteria. The most stricking difference is the presence on the fracture faces of the LDC cell wall of different classes of particulated entities of yet unknown nature. In the mycobacteria and LDC the periseptal annuli likely provide a potential frame for cell envelope and cell membrane assembly.

Animals↗

PubMed1982

The structure of the cytoplasmic matrix preserved by freeze-drying and freeze-substitution.

This paper reports a study of the cytoplasmic matrix in whole cultured cells examined by high voltage electron microscopy. In order to acquaint ourselves with the influence of preparation procedures on the morphology depicted, PtK2 cells were prepared for examination by first freezing in propane at -185 degrees C and then drying while maintained at -95 degrees C. Other cells of the same type were first fixed with glutaraldehyde and OsO4 and then frozen dried. Others were prepared by freeze-substitution and eventually dried by the critical-point method from CO2. And finally, some were preserved by conventional techniques of glutaraldehyde and OsO4 followed by dehydration in alcohol and critical-point drying. The observations are presented in stereo images. The morphologies after these various procedures are quite similar. All show the characteristic three-dimensional lattice or meshwork of slender filaments called microtrabeculae. In cells rapidly frozen and then dried from the frozen state, there is less evidence of shrinkage and probable change in the trabecular structure than in cells first fixed with glutaraldehyde and then frozen dried. The differences we relate to the demonstrable failure of glutaraldehyde to penetrate the cell quickly and fix instantly any component that is in active motion. Other differences that can be observed between all four types of preparation are not recognizably striking and are thought to reflect as much as anything morphological diversity in the original cells. In some instances, the amorphous ice of the initial freezing at -185 degrees C was allowed to crystallize at -80 degrees C. The small crystals that form push aside the microtrabeculae and leave obvious imprints on the structure. The essential message from these experiments is that the cytomatrix is structured.

Animals↗

PubMed1995

Comparison of ultrastructure of germinating pea leaves prepared by high-pressure freezing-freeze substitution and conventional chemical fixation.

High-pressure freezing-freeze substitution (HPF-FS) methods were applied to germinating pea leaves. Good ultrastructural preservation without visible freezing damage was obtained up to 200 microns in thickness. Compared to conventional chemical fixation (CF), cellular membranes were smoother without undulation, and organelles appeared more turgid. The matrices of cytoplasm and organelles were denser and more homogeneous. The features imply that HPF-FS samples retain more substances and ultrastructure closer to the living state. There were differences in membrane stainability among organelles in HPF-FS specimens, which were not seen after CF. Bundles of microfilaments were observed frequently after HPF-FS.

Freeze Substitution↗

PubMed1997

Reevaluation of the effect of lysoyzme on Escherichia coli employing ultrarapid freezing followed by cryoelectronmicroscopy or freeze substitution.

Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer. Gram-negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180-187] found that lysozyme per se is able to kill some Gram-negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.

Escherichia coli↗

PubMed1992

Intercellular junctions in embryonic chick cardiac muscle revealed by rapid freezing and freeze-substitution.

Using the method of rapid freezing and freeze-substitution, the embryonic chick cardiac muscle was investigated by transmission electron microscopy. Initially, the intercellular junctional complexes (fasciae adherentes and desmosomes) were formed in close proximity to each other along a nearly straight line. Subsequently, the separation of fasciae from desmosomes took place to form intercalated discs. The cell membranes of fasciae adherentes were reinforced with highly interwoven fine fibrils at which myofibrils terminated. The intercellular space of fasciae was bridged with fine fibrillar structures seemingly connected by a thin line at their middle portions. In the intercellular space of desmosomes, central lamina and traversing filaments were clearly observed. The outer and inner leaflets of the desmosomal plasmalemma were asymmetrically differentiated; the outer leaflet was thinner than the inner leaflet. On the inner side of the cell membrane, an electron-lucent layer and a dense desmosomal plaque were observed. The latter structure had protrusions with less electron density towards the cytoplasmic side. Further inside, a meshwork of fine fibrils was seen along and toward which bundles of intermediate filaments ran. The results obtained with freeze-substitution appeared to provide more information than those with thin sections after conventional fixation or with replicas of chemically fixed/glycerinated or physically fixed/deep-etched materials.

Animals↗

PubMed1993

Improved preservation of ultrastructure in difficult-to-fix organisms by high pressure freezing and freeze substitution: I. Drosophila melanogaster and Strongylocentrotus purpuratus embryos.

In this study, we have applied the techniques of high pressure freezing and freeze substitution to embryonic cell types which are usually difficult to fix properly for electron microscopy. In both Drosophila and Strongylocentrotus purpuratus, we see improved preservation of both membrane systems and cytoskeleton when compared to published results on the same cells using conventional electron microscope (EM) fixation methods. Finally, we have seen that postembedding labelling of sections is possible even after light osmium fixation during freeze substitution.

Animals↗

PubMed1999

Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose. I. Experimental measures.

In this study, two methods are used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing in the presence and absence of a cryoprotectant (glucose). The first is a "two-step" low-temperature microscopy (equilibrium and dynamic) freezing method and the second is a differential scanning calorimeter (DSC) technique. These two techniques were used to study (i) the in vitro architecture of R. sylvatica frog liver tissue and to measure its characteristic Krogh cylinder dimensions; (ii) the "equilibrium" (infinitely slow) cooling behavior and the osmotically inactive cell volume (V(b)) of R. sylvatica liver cells; and (iii) the dynamic water transport response of R. sylvatica liver cells in the presence and absence of the CPA (glucose) at a cooling rate of 5 degrees C/min. Stereological analysis of the slam frozen (>1000 degrees C/min) micrographs led to the determination that 74% of the liver tissue in control frogs was cellular versus 26% that was extracellular (vascular or interstitial). Mapping the stereological measurements onto a standard Krogh cylinder geometry (Model 1) yielded distance between adjacent sinusoid centers, DeltaX = 64 microm; original sinusoid (vascular) radius, r(vo) = 18.4 microm; and length of the Krogh cylinder, L = 0.71 microm (based on an isolated frog hepatocyte cell diameter of 16 microm). A significant observation was that approximately 24% of the frog hepatocyte cells are not in direct contact with the vasculature. To account for the cell-cell contact in the frog liver architecture a modified Krogh cylinder geometry (Model 2) was constructed. In this model (Model 2) a second radius, r(2) = 28.7 microm, was defined (in addition to the original sinusoid radius, r(vo) = 18.4 microm, defined above) as the radius of the membrane between the adjacent cells (directly adjacent to vascular spaces) and embedded cells (removed from vascular spaces). By plotting the two-step equilibrium cooling results on a Boyle-van't Hoff plot, the osmotically inactive cell volume, V(b) was obtained as 0.4. V(o) (where V(o) is the isotonic cell volume). The two-step dynamic micrographs and the heat release measurements from the DSC were used to obtain water transport data during freezing. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min.

Animals↗

PubMed1999

Liver freezing response of the freeze-tolerant wood frog, Rana sylvatica, in the presence and absence of glucose. II. Mathematical modeling.

The "two-step" low-temperature microscopy (equilibrium and dynamic) freezing methods and a differential scanning calorimetry (DSC) technique were used to assess the equilibrium and dynamic cell volumes in Rana sylvatica liver tissue during freezing, in Part I of this study. In this study, the experimentally determined dynamic water transport data are curve fit to a model of water transport using a standard Krogh cylinder geometry (Model 1) to predict the biophysical parameters of water transport: L(pg) = 1.76 microm/min-atm and E(L(p)) = 75.5 kcal/mol for control liver cells and L(pg)[cpa] = 1.18 microm/min-atm and E(L(p))[cpa] = 69.0 kcal/mol for liver cells equilibrated with 0.4 M glucose. The DSC technique confirmed that R. sylvatica cells in control liver tissue do not dehydrate completely when cooled at 5 degrees C/min but do so when cooled at 2 degrees C/min. Cells also retained twice as much intracellular fluid in the presence of 0.4 M glucose than in control tissue when cooled at 5 degrees C/min. The ability of R. sylvatica liver cells to retain water during fast cooling (>/=5 degrees C/min) appears to be primarily due to its liver tissue architecture and not to a dramatically lower permeability to water, in comparison to mammalian (rat) liver cells which do dehydrate completely when cooled at 5 degrees C/min. A modified Krogh model (Model 2) was constructed to account for the cell-cell contact in frog liver architecture. Using the same biophysical permeability parameters obtained with Model 1, the modified Krogh model (Model 2) is used in this study to qualitatively explain the experimentally measured water retention in some cells during freezing on the basis of different volumetric responses by cells directly adjacent to vascular space versus cells at least one cell removed from the vascular space. However, at much slower cooling rates (1-2 degrees C/h) experienced by the frog in nature, the deciding factor in water retention is the presence of glucose and the maintenance of a sufficiently high subzero temperature (>/=-8 degrees C).

Animals↗

PubMed1987

Ultrastructural observations on chick bone processed by quick-freezing and freeze-substitution.

The ultrastructure of newly formed bone was examined with the use of quick-freezing followed by freeze-substitution. Osteoblasts and young osteocytes were characterized by a smooth cell contour, whereas old osteocytes were irregular in shape. The plasma and intracytoplasmic membranes were clearly identifiable as trilaminar substructures. With the method described herein the tissue is handled in the anhydrous state. Thus mitochondrial granules could be demonstrated in all samples, since their preservation is not affected by non-aqueous solutions. The matrices of intact mitochondria were densely stained with post-staining. The contents of the Golgi complex, rough-surfaced endoplasmic reticulum (RER), nuclear envelope, vesicles, and vacuoles were stained to various degrees. Lacunar spaces were always filled with flocculent and filamentous materials, and the plasma membrane was in direct contact with them. Membrane-bounded matrix vesicles were clearly visible within the osteoid extracellular matrix which was the initial site of mineral crystal deposition. In heavily mineralized bone matrix, the periodic pattern of collagen fibrils was retained, and the electron density of mineralized matrix in freeze-substituted and unstained sections which had been floated on ethylene glycol was greater than that encountered in sections processed in aqueous reagents.

Animals↗

PubMed1989

Quantitative X-ray microanalysis of calcium in sea urchin eggs after quick-freezing and freeze-substitution. Validity of the method.

Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species (Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2 +/- 0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3 +/- 1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of 45Ca from prelabelled eggs. Exchangeable 45Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles.

Animals↗

PubMed1994

Ultrastructure of guinea pig stria vascularis processed by rapid freezing and freeze substitution.

The rapid-freezing and freeze-substitution method fixes a specimen as if it were prepared before excision. We used this method to study the stria vascularis of guinea pigs using electron microscopy. Findings were essentially the same as those obtained with conventional chemical fixation, although freeze substitution made it possible to observe the membrane structures in a smoother and more linear manner. This method seems to be the procedure of choice for studying the instantaneous movement and behavior of cells.

Animals↗

PubMed1987

A comparative study of mouse embryo freeze-preservation including the examination of a thermoelectric freezing device.

In Study 1 over 2000 4- to 8-cell mouse embryos were randomly pooled and assigned to 1 of 12 treatment groups. A 2 X 2 X 3 factorial design was used to analyze two types of cryoprotectant/post-thaw (PT) dilutions (dimethyl sulfoxide [Me2SO]/stepwise dilution versus glycerol/sucrose dilution), two storage containers (glass ampoules versus plastic straws), and three cooling treatments. Two commercial, controlled-rate freezing machines were examined, employing either nitrogen gas (Planer) or thermoelectric (Glacier) cooling. Embryos were cooled slowly (0.5 degrees C/min) to -35 or -80 degrees C and then cooled rapidly by transfer into liquid nitrogen (LN2). Thawed embryos were cultured for 24 hr after which developmental stage, post-thaw survival (PTS), embryo degeneration rate (EDR), quality grade (QG), and fluorescein diacetate viability grade (VG) were assessed. Overall, PTS and EDR were similar (P greater than 0.05) among the three freezing unit/plunge temperature treatments. Cumulative results of container and cryoprotectant/PT dilution treatments consistently demonstrated greater PTS, QG, and VG ratings and lower EDR values when embryos were frozen in ampoules using glycerol/sucrose dilution. Embryos treated with Me2SO/stepwise dilution were particularly sensitive to freezing damage when stored in plastic straws and plunged into LN2 at -35 degrees C. Study 2 was directed at determining whether Study 1 methods for diluting Me2SO-protected embryos markedly affected PTS rates. Post-thaw culture percentages were no different (P greater than 0.05) for four- to eight-cell Me2SO-treated embryos frozen in ampoules (using the forced-LN2 device), thawed, and diluted either conventionally in reduced concentrations of Me2SO or in the sucrose treatment normally accorded glycerolated embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

The use of high pressure freezing and freeze substitution to study host-pathogen interactions in fungal diseases of plants.

This article reports on the use of high pressure freezing followed by freeze substitution (HPF/FS) to study ultrastructural details of host-pathogen interactions in fungal diseases of plants. The specific host-pathogen systems discussed here include a powdery mildew infection of poinsettia and rust infections of daylily and Indian strawberry. The three pathogens considered here all attack the leaves of their hosts and produce specialized hyphal branches known as haustoria that invade individual host cells without killing them. We found that HPF/FS provided excellent preservation of both haustoria and host cells for all three host-pathogen systems. Preservation of fungal and host cell membranes was particularly good and greatly facilitated the detailed study of host-pathogen interfaces. In some instances, HPF/FS provided information that was not available in samples prepared for study using conventional chemical fixation. On the other hand, we did encounter various problems associated with the use of HPF/FS. Examples included freeze damage of samples, inconsistency of fixation in different samples, separation of plant cell cytoplasm from cell walls, breakage of cell walls and membranes, and splitting of thin sections. However, we believe that the outstanding preservation of ultrastructural details afforded by HPF/FS significantly outweighs these problems and we highly recommend the use of this fixation protocol for future studies of fungal host-plant interactions.

Ascomycota↗

PubMed2001

Acanthamoeba castellanii: ultrastructure of trophozoites using fast freeze-fixation followed by freeze-substitution.

In recent years, the use of fast-freeze fixation followed by freeze-substitution has been shown to be the procedure that best satisfies the ultrastructural preservation of cellular components due to the rapidity of the fixation procedure and the reduction of artifacts compared to chemical fixation. When these techniques were used to study the fine structure of axenically cultured trophozoites of Acantamoeba castellanii, an improved preservation of the whole cell was observed. The ground substance of the cytoplasm is densely packed with fibrogranular material which is frequently removed with the use of conventional techniques. Also, the ultrarapid physical stabilization allows the visualization of fusion and fission processes of cytoplasmic vacuoles and vesicles. The nuclear structure and cytoplasmic microfilaments as well as membranous structures were clearly identified. Low temperature techniques combine the advantage of fast-freeze fixation for the physical stabilization of organic molecules and their stabilization by the substitution medium at low temperature giving rise not only to a better preservation of the cell ultrastructure but providing a favorable basis for immunocytochemistry at the electron microscopy level.

Acanthamoeba↗

PubMed1990

The effects of freezing or freeze-drying on the biomechanical properties of the canine intervertebral disc.

The transplantation of spinal allografts to correct defects that include disc, body, or segments of both is currently under experimental investigation. A method of graft preservation that will least compromise the biomechanical integrity of the bodies or discs is required. Using a five-mode biomechanical analysis, the authors compared the change in stiffness of ten preserved canine spines. The freeze-dried specimens lost a significantly greater amount of stiffness in compression, flexion, extension, and torsion than did the frozen specimens. Therefore, from a biomechanical viewpoint, deep-freezing is superior to freeze-drying for spinal allograft preservation.

Animals↗

PubMed1998

Biophysics of freezing in liver of the freeze-tolerant wood frog, R. sylvatica.

This study investigates the water transport characteristics during freezing in the liver tissue of the freeze-tolerant wood frog Rana sylvatica. Experiments were performed using both low temperature microscopy and a differential scanning calorimeter (DSC). Tissue samples were cooled at 2 and 5 degree C/min by a "two-step" freezing technique to end temperatures of -4, -6, -8, -10, and -20 degrees C, followed by a slam cooling (> 1000 degrees C/min) step. Stereological analysis of the low temperature microscopy results leads to the conclusions that 74% of the control tissue is cellular (26% vascular), Vb (osmotically inactive cell volume) is 0.4 Vo and the Krogh cylinder dimensions are: distance between adjacent sinusoid centers, delta X = 64 microns, original sinusoid (vascular) radius, rvo = 18.4 microns and length of the Krogh cylinder, L = 0.71 microns (assuming a single isolated hepatocyte cell diameter of 16 microns). A parallel study was also done using the DSC at 2 and 5 degrees C/min, and the measured heat releases from the tissue were used to calculate water transport data. Both techniques confirmed that tissue cooled at 5 degrees C/min does not dehydrate completely, but does so when cooled at 2 degrees C/min. By curve fitting a model to 5 degrees C/min water transport data from both techniques the biophysical parameters of water transport were obtained: Lpg = 1.76 microns/min-atm and ELp = 75.5 Kcal/mol. A modified Krogh model was used to account for the fact that approximately 24% of the hepatocytes were found not to be in direct contact with the vasculature. This model was then used to explain the experimentally measured water retention in some cells on the basis of different volumetric responses to dehydration of cells directly adjacent to vascular spaces and cells at least one cell removed from the vascular spaces.

Animals↗

PubMed1973

Envelope of mouse mammary tumor virus studied by freeze-etching and freeze-fracture techniques.

As part of a study of the cell surface changes associated with the production of murine mammary tumor virus, the structure of the envelope of this virus has been examined by using freeze-fracture techniques. Both fracture and deep-etch surfaces were examined. The fracture faces contain 10-nm spheres comparable to those observed on fractured plasma membranes, although fewer in number. Surfaces exposed by etching possess a highly regular hexagonal array of pits 25 nm apart. By examining freeze-fracture and freeze-etch preparations of virus with ferritin covalently bound to its surface, it has been determined that the surface exposed by etching is the outer surface of the virus. The pitted exterior surface of the mammary tumor virus appears to be a unique surface structure.

Dimethyl Sulfoxide↗