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PubMedDate not supplied

Freeze substitution and freeze drying for stable, long-term preservation of cytologic specimens for immunostaining.

OBJECTIVE: To develop a new method of fixing and preserving cytologic specimens for immunostaining after long-term storage at room temperature. STUDY DESIGN: The method consists of three steps: fixation, freeze substitution or freeze drying, and storage. To test the method, we used the human small cell lung carcinoma cell line Lu-135, which expresses a high level of mutant p53 protein and exhibits strong nuclear staining when reacted with an anti-p53 antibody. A smear of Lu-135 cells was fixed in a mixture of methanol and ether (50:50=vol/vol) or sprayed with a fixative containing isopropyl alcohol, methanol and polyethylene glycol. The fixed cells were freeze substituted by immersing them in a dry ice/methanol/ether bath and then were freeze dried under a vacuum. The smear was then placed in a 50-mL conical tube containing silica gel. The tube was sealed and stored at room temperature. RESULTS: Freeze substituted cells that were fixed with methanol/ether and stored for more than six months retained strong p53 positivity, as strong as that of the control cells, which had been fixed and stored in methanol. CONCLUSION: Freeze substitution and freeze drying are an alternative method of preserving cytologic specimens.

Carcinoma, Small Cell↗
PubMed1993

Cryofixation of basement membranes followed by freeze substitution or freeze drying demonstrates that they are composed of a tridimensional network of irregular cords.

Since conventional chemical fixation may extract tissue components and thus alter structural organization, cryofixation was used to reexamine the ultrastructure of three thick basement membranes: lens capsule, Reichert's membrane, and Engelbreth-Holm-Swarm (EHS) tumor matrix, and two thin basement membranes, those of epididymis and semi-niferous tubules. Cryofixation was achieved by slam freezing followed by either freeze substitution in dry acetone containing 1% osmium tetroxide and 0.05% uranyl acetate or freeze drying in a molecular distillation dryer. The results by both procedures demonstrate that thick basement membranes and the lamina densa of thin basement membranes are composed of a network of anastomosing strands referred to as cords. The cords vary in density and distinctiveness, but their thickness averages 3 to 5 nm in every tissue examined. The spaces separating the cords vary within wide limits, but their mean diameter is approximately 15 nm in every case. Two other common features are 1) the presence within the network of a few 1.5-3.0-nm-thick filaments and 2) 4.5-nm-wide sets of parallel lines referred to as double tracks. When these results are compared with those previously described after conventional fixation, no significant difference is observed in either the cord network or the associated filaments and "double tracks." However, in the thin basement membranes processed by cryofixation, the lamina densa is in direct contact with epithelial cells, whereas, after conventional fixation, the lamina densa is separated from the epithelial cells by a pale layer referred to as lamina lucida or lamina rara. Immunogold labeling of three basement membranes after cryofixation and freeze substitution in acetone containing 0.3% glutaraldehyde yields strong reactions for laminin, type IV collagen, and heparan sulfate proteoglycan. Comparison with previous results indicates that conventional formaldehyde fixation adequately preserves laminin and type IV collagen but causes the loss of some proteoglycan. It is concluded that, except for this loss and the absence of lamina lucida in cryofixed thin basement membranes, the morphological and antigenic features obtained after cryofixation are similar to those observed in the past after conventional fixation.

Animals↗
PubMed1994

A new versatile system for freeze-substitution, freeze-drying and low temperature embedding of biological specimens.

A universal system for freeze-substitution (FS), freeze-drying (FD) and low temperature embedding (LTE) has been developed, suited to perform standardized procedures of cryoprocessing biological and medical specimens as well as systematic studies of dehydration and embedding at various low and high temperatures. In a 35 1 Dewar vessel with 110 mm neck diameter an aluminum tube is mounted to the bottom of the liquid nitrogen (LN2x) reservoir and extends to the lower part of the cylindrical neck. At its top an aluminum plate serves as a contact surface for either the FS chamber or the FD chamber. Fs and subsequent LTE are carried out in an environment of dry cold nitrogen gas provided by evaporating nitrogen from the dewar. Different capsules and moulds may be used for cryodehydration and LTE. FD of bulk specimens or cryosections takes place in an absolutely clean vacuum provided by a cryosorption pump integrated in the FD apparatus. Most of the H2O molecules from the frozen specimen are trapped by large cold surfaces inside the drying chamber. Due to the low LN2 consumption during FS or FD (3-4 1 LN2/day) both procedures may be carried out for 8-10 days without refilling the dewar. A few representative results show that well frozen biological material is stabilized by prolonged FS or FD at temperatures of about -80 degrees C without user of chemical fixatives like OsO4 in the substitution medium during FS or by OsO4 vapor fixation after FD.

Animals↗
PubMed1980

Cryofixation without cryoprotectants. Freeze substitution and freeze etching of an insect olfactory receptor.

Antennae of the silk moth, Bombyx mori, were frozen by immersion into propane at -180 degrees C, and further processed by (a) freeze substitution (FS) or (b) freeze etching (FE). Although no cryoprotectant was used, freezing damage was observed in deeper tissue regions only. Data from FS specimens closely resemble those from FE replicas. Therefore, FS usually does not induce noticeable secondary artefacts by the preparation steps subsequent to freezing. When compared with chemically fixed antennae, the superior quality of cryofixation in this tissue is evident, particularly where cell surfaces and processes border the receptor lymph cavity; membranes are smooth following a steady course; dendrites and axons are round in cross-section with evenly distributed microtubules. The value of cryofixation is discussed with special reference to structures of presumed functional significance (e.g. stimulus conducting pore tubules, intramembrane particles of the receptor membrane, the ciliary segment of the dendrites, intercellular dilations, membrane junctions).

Animals↗
PubMed1984

Ciliated and microvillous structures of rat olfactory and nasal respiratory epithelia. A study using ultra-rapid cryo-fixation followed by freeze-substitution or freeze-etching.

The olfactory epithelium of the Sprague-Dawley rat showed structures which indicate that freeze-substitution after ultra-rapid cryo-fixation is a better method for its preservation than conventional fixation techniques. A new feature is that matrices of the distal parts of olfactory cilia range in their staining intensity from very dense to electron-lucent. Outlines of structures are smooth and membrane features can be clearly seen. The textures of mucus from olfactory and respiratory epithelia are distinctly different after freeze-fracturing and deep-etching following cryo-fixation. Olfactory cilia show no microtubule-attached axonemal structures. Cross-sectional diameters are smaller after freeze-substitution than after freeze-fracturing. Intramembranous particle densities are lower in nine regions of three cell types in cryo-fixed olfactory and respiratory epithelia than in those chemically fixed and cryoprotected. The fracture faces of membranes from etched, cryo-fixed cells have holes, a result which probably accounts for differences in particle density between cryo-fixed and chemically-fixed, cryo-protected cells. Particle diameters are usually the same using both methods. Densities of intramembranous particles and particles plus holes are highest in supporting cell processes, followed by endings and cilia of olfactory receptor cells, and are lowest in respiratory cilia. Particle densities at outer and inner surfaces are higher than those in either fracture face. Outer surfaces show a good correlation from region to region with densities summated over both fracture faces.

Animals↗
PubMed1958

Methods and principles of fixation by freeze-substitution.

Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O degrees C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175 degrees C. (with precautions). The frozen tissue was placed in substituting fluid at -70 degrees C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances.

Acetone↗
PubMed1982

Freeze-substitution and isothermal freeze-fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (-21 degrees C).

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethylsulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze-substitution and isothermal freeze-fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze-substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configuration at this relatively high substitution temperature. The results of freeze-substitution in dimethylsulphoxide were confirmed by isothermal freeze-fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0.3 K min-1 or 2 K min-1 were related to the distribution of the ice phase within the tissue.

Animals↗
PubMed1982

Freeze-substitution.

Freeze-substitution is a technique suitable for the preparation of unicellular and multi-cellular plant and animal specimens for conventional light microscopy, TEM and SEM. It is also widely used as a means of preparing animal and plant tissues for the localization of water soluble substances by analytical electron microscopy, autoradiography or visual detection of precipitates. The technical requirements of preparation, together with an evaluation of the procedures, are presented for various applications. Careful selection and evaluation of freezing technique, substitution solvent and regime are required for meaningful results.

Animals↗
PubMed1993

Freeze substitution after fast-freeze fixation in preparation for immunocytochemistry.

As compared to classical chemical fixation, the physical immobilization of ultrastructures by fast-freeze fixation (FFF) and the subsequent exchange of water in its solid state by freeze substitution (FS) improve the preparation procedure for immunogold labeling (IGL). FFF-FS results in a morphological preservation of unchallenged quality, as well as in a better preservation of antigenic reactivity, thus allowing remarkable precision of labeling on sections. However, FFF, particularly over a cooled metal plate, requires a heavy and expensive machine. It is not suitable for all biological specimens and in the best conditions, which remain difficult to standardize, the thickness of the well-preserved portion of the specimen does not exceed a few microns for compact tissues, and exceptionally 30-40 microns for isolated cells. The FS procedure is long and must be adjusted empirically for every new specimen and antigenic detection. The preservation of a given antigen's reactivity in the presence of fixative agents and embedding resins remains unpredictable. The action of fixative agents is different and milder in FS than when they are used classically in chemical fixation. By chance, one of the best FS procedures for the preservation of both ultrastructure and antigenicity appears to be by using acetone alone, together with a molecular sieve to improve the water exchange process. A large choice of embedding resins usually allows us to find a compromise between ultrastructural and antigenic preservation.

Animals↗
PubMed1990

Preparation of the nematode-trapping fungus, Arthrobotrys oligospora, for scanning electron microscopy by freeze substitution.

A freeze-substitution technique for preparing fungal specimens for scanning electron microscopy is described. This involves cryofixation in liquid nitrogen, freeze substitution in methanol at -20 degrees C and critical-point drying. The trapping complexes and conidiophores of the nematophagous fungus Arthrobotrys oligospora are well preserved and retain their normal three-dimensional arrangement.

Animals↗
PubMed1991

Routine cryofixation of plant tissue by propane jet freezing for freeze substitution.

Cryofixation and freeze substitution methods were developed for ultrastructural studies of cells in complex plant tissues. Leaf tissues and root tips of tobacco (Nicotiana tabacum L. var. Maryland Mammoth) were frozen with a RMC MF7200 propane jet freezer and freeze substituted sequentially with tannic acid and osmium tetroxide/uranyl acetate in acetone. High quality preservation was consistently obtained for epidermal and phloem cells of the leaf, and epidermal, cortical, meristematic, and cap cells of the root tip. Leaf mesophyll cells were also often well frozen. Organelles, including nuclei, endoplasmic reticulum, mitochondria, Golgi bodies, and plastids, showed excellent structural integrity and contrast. Most notable is the superior preservation of the cytoskeleton. Our results demonstrate that the propane jet freezer can be used routinely for high quality cryofixation of higher plant cells in certain complex tissues. This could have important implications for the use of cryofixation approach in a wide range of research in plant biology.

Cryopreservation↗
PubMed1993

Comparison of the ability of freeze etch and freeze substitution to preserve actin filament structure.

In order to test the ability of freeze substitution to accurately preserve the ultrastructure of the actin component of the cytoskeleton, the structure of rotary shadowed actin filaments was compared following preparation by glutaraldehyde fixation and freeze etch or freeze substitution. Freeze substituted actin filaments were further processed by either etching away frozen organic solvent or critical-point-drying before rotary shadowing. Comparison of filament diameters showed no significant difference between actin filaments that were directly etched and those that were freeze substituted and then etched. However, freeze substituted and then critical-point-dried filaments were significantly larger in diameter than filaments that were directly etched in water. The long pitch (right-handed) two start helix was not affected by the different methods of preparation. However, the left-handed "genetic" helical repeat that was prominent in actin filaments prepared by freeze etch was more difficult to detect in freeze substituted specimen, especially following critical-point-drying. Although the organization and distribution of actin filaments in extracted cells was similar in both freeze substituted and freeze etched specimens, there were some detectable differences. In cells that were freeze substituted and then critical-point-dried, filaments appeared to intersect at greater angles and seemed more "taut." These results suggest that freeze substitution can preserve the overall morphology of actin filaments, but some chemical or physical modification of macromolecular surface structure may occur during the substitution process and these changes may be further exaggerated by subsequent processing steps.

Actins↗
PubMed2001

Freeze-substitution: origins and applications.

Freeze-substitution is a physicochemical process in which biological specimens are immobilized and stabilized for microscopy. Water frozen within cells is replaced by organic solvents at subzero temperatures. Freeze-substitution is widely used for ultrastructural and immunocytochemical analyses of cells by transmission and scanning electron microscopy. Less well recognized is its superiority over conventional chemical fixation in preserving labile and rare tissue antigens for immunocytochemistry by light microscopy. In the postgenome era, the focus of molecular genetics will shift from analyzing DNA sequence structure to elucidating the function of gene networks, the intercellular effects of polygenetic diseases, and the conformational rearrangements of proteins in situ. Novel strategies will be needed to integrate knowledge of chemical structures of normal and abnormal macromolecules with the physiology and developmental biology of cells and tissues from whole organisms. This review summarizes the progress and future prospects of freeze-substitution for such explorations.

Animals↗
PubMed1991

Freeze-substitution and the preservation of diffusible ions.

Freeze-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 (T1+) of T1(+)-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray microanalytical 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 macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.

Acetone↗
PubMed2002

Electron microscopic studies of three gliding Mycoplasmas, Mycoplasma mobile, M. pneumoniae, and M. gallisepticum, by using the freeze-substitution technique.

Freeze-substitution technique was applied to thin-sectioning electron microscopy of Mycoplasma mobile, M. pneumoniae, and M. gallisepticum, all of which can glide in the direction of the tapered cell end. M. mobile presented a flask-like cell morphology. An additional layer was found around the tapered end. The cell images of M. pneumoniae showed a protruding membrane extension, the attachment organelle, composed of a low density space inside the cells and featuring a filamentous dense core anchored to the terminal end. The detailed structures were more obvious than those observed by the conventional chemical fixation. The cells of M. gallisepticum presented irregular dense granules, in contrast to regular particles, which can be observed in the images of chemically fixed thin sections, in the rear portion of the cells.

Freezing↗
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↗
PubMed1972

The Retention of Water-soluble Compounds during Freeze-Substitution and Microautoradiography.

Freeze-substitution and Epon embedment were quantitatively evaluated for their effectiveness in retaining water-soluble metabolites in plant tissues. Roughly 99% of the 80% (v/v) ethanol-extractable radioactivity in photosynthetically labeled soybean leaf discs and in petiole fragments containing translocated (14)C was retained during freeze-substitution in acetone or propylene oxide and embedment in Epon. Substantially more activity was lost from (14)C-sucrose-infiltrated pith blocks, but most or all of this loss came from the block surface. The procedure was effective for a sucrose concentration as low as 0.004%. Sections floated on water retained most of their (14)C-sucrose, and high resolution autoradiographs could easily be prepared without resorting to dry procedures. Embedded (14)C-sucrose was apparently chemically unreactive, since there was no loss of radioactivity when sections were stained with the periodic acid-Schiff reagent, nor did the embedded sucrose show staining.

Journal Article↗