scholarly journals Methods and Principles of Fixation by Freeze-Substitution

1958 ◽  
Vol 4 (5) ◽  
pp. 593-602 ◽  
Author(s):  
Ned Feder ◽  
Richard L. Sidman
Keyword(s):  
Low Temperatures ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Picric Acid ◽  
Freeze Substitution ◽  
Tissue Structure ◽  
Chemical Agent ◽  
Rapid Freezing ◽  
Chemical Mechanisms ◽  
Accurate Localization

Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O°C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175°C. (with precautions). The frozen tissue was placed in substituting fluid at -70°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.

Development of spermatia in the red alga Caloglossa leprieurii preserved for TEM by freeze substitution

1990 ◽  
Vol 48 (3) ◽  
pp. 668-669
Author(s):  
Wilma L. Lingle ◽  
David Porter ◽  
Marshall Darley
Keyword(s):  
Red Algae ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Red Alga ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Liquid Propane ◽  
Laboratory Cultures ◽  
To Come

The red algae have been difficult to preserve for TEM observation. In an effort to overcome the limitations of conventional, aqueous fixations, we used rapid freezing techniques that are superior in preserving delicate and transient membrane features. Laboratory cultures of the red alga Caloglossa leprieurii were prepared for TEM by rapid freezing in liquid propane followed by substitution at -80° in 2% osmium tetroxide and 0.05% uranyl acetate in dry acetone for 65 hours. While in substitution fluid, the tissue was allowed to come to room temperature over a 6 hour period. The fluidwas replaced with dry acetone, followed by two 30 minute washes. Infiltration with Embed 812 without accelerator was performed at 4° on a tumbler in increments of 12.5% changed every 12 hours. Five percent increments were utilized from 75% to 100%. Three 24 hour exchanges with 100% resin including accelerator took place alternately under vacuum at room temperature for 8 hours, then at 4°on a tumbler for 16 hours. The tissue was cured at 60°.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

Freeze substitution studies on cultured human fibroblasts

1989 ◽  
Vol 47 ◽  
pp. 1008-1009
Author(s):  
Julian P. Heath ◽  
Donna Turner
Keyword(s):  
Osmium Tetroxide ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Lung Fibroblasts ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Cultured Human Fibroblasts

We are using rapid freezing and freeze substitution to study the three dimensional organisation of membrane systems and cytoskeletal filaments in motile fibroblasts. This study has two objectives: first, to provide material for structural and immunocytochemical analysis of membrane-cytoskeletal interactions in cells that have been preserved with minimum artefact (1,2,3) and second, to refine and develop existing rapid freezing and freeze substitution techniques to allow for the study of single cells that have been experimentally manipulated and observed by digital video microscopy before fixation.The cells used were human lung fibroblasts (IMR90) either growing on Lux Thermanox coverslips or as pelleted suspensions. The cells were slam frozen on a Med-Vac Cryo Press against a liquid nitrogen cooled copper block. Coverslips were trimmed to 2 x 2 mm in size, excess fluid was drained off, and they were placed on top of a 1mm thick gelatin cushion on an aluminium planchette. For cell suspensions, 3 ul was placed on top of the gelatin cushion. Frozen samples were placed in acetone containing 1% osmium tetroxide for 72 hours at 192 K, wanned to 253 K for 4 hours, and then brought to room temperature. The samples were rinsed in acetone and embedded in Spurr’s resin. Thin sections were cut on a RMC6000 ultramicrotome, stained in uranyl acetate and Reynolds' lead citrate and photographed on a Philips EM410 electron microscope at 60 keV.

scholarly journals Rapid-freezing and malachite green-acrolein-osmium tetroxide freeze-substitution fixation improve visualization of extracellular lipids in rat incisor pre-dentin and dentin.

1987 ◽  
Vol 35 (4) ◽  
pp. 427-433 ◽  
Author(s):  
M Goldberg ◽  
F Escaig
Keyword(s):  
Malachite Green ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Dense Network ◽  
Rat Incisor ◽  
Unequal Distribution ◽  
Tissue Sections ◽  
Extracellular Matrix Components ◽  
Matrix Components

Rat incisor tissue sections were fixed by a modified version of the malachite green-aldehyde method (MGA) composed of rapid-freezing, malachite green-acrolein staining, and osmium tetroxide freeze-substitution (Fr.MGAO). In the pre-dentin, a thick, dense network of branched fibrous structures was observed. Cryotechniques allowed visualization of complexes about twice as thick and dense as the aggregates visualized on MGA-treated sections. Pretreatment of rapid-frozen samples with methanol before freeze-substitution fixation and staining prevented staining of the complexes otherwise revealed by the Fr.MGAO method. Electron-dense material stained by this procedure resisted de-mineralization with EDTA, while intramitochondrial granules and dentin crystallites were dissolved. EDTA treatment demonstrated unequal distribution of Fr.MGAO staining in dentin in the form of tiny dots underlining the collagen fibers. These results support the concept that rapid-freezing, followed by staining and freeze-substitution fixation, improves preservation of the phospholipids visualized as extracellular matrix components in pre-dentin and dentin of rat incisors.

A New Smaller Sized Virus-Like Particle in Drosophila Melanogaster

2001 ◽  
Vol 7 (S2) ◽  
pp. 742-743
Author(s):  
Jeffrey G. Ault ◽  
Ellen Shimakawa
Keyword(s):  
Drosophila Melanogaster ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
High Pressure Freezing ◽  
Chromosome Behavior ◽  
Thin Sections ◽  
Behavior Study ◽  
Virus Like Particle

During a chromosome behavior study involving high-pressure freezing (HPF)/freeze substitution (FS) of Drosophila melanogaster testes, we discovered quasi-crystalline inclusions in the nuclei of adjacent gut epithelial cells (Fig. 1). The HPF and FS protocols were standard. The viscera of adult flies were packed in yeast paste for HPF. The tissue was fixed by FS with 1% osmium tetroxide in acetone for 72 hours at -90° C then 48 hours at -60° C. Afterwards, it was washed several times at room temperature in 100% acetone and embedded in Epon/Araldite. Thin sections were cut and stained with uranyl acetate and lead. As expected with HPF/FS, the material was well-preserved with straight microtubules, smooth membranes, dense mitochondria, and abundant ribosomes both on the rough endoplasmic reticulum and in the full cytoplasm (Fig. 1).The inclusions consisted of virus-like particles packed loosely together in orderly arrays. Particles were usually hexagonally packed with spaces disrupting the periodicity (Figs. 2 and 3).

The Ultrastructure of Myocardial Cells in Normal and Cardiac Lethal Mutant Mexican Axolotls, (Ambystoma Mexicanum)

1970 ◽  
Vol 28 ◽  
pp. 62-63
Author(s):  
Larry F. Lemanski ◽  
Eldridge M. Bertke ◽  
J. T. Justus
Keyword(s):  
Fine Structure ◽  
Heart Development ◽  
Osmium Tetroxide ◽  
Picric Acid ◽  
Ambystoma Mexicanum ◽  
Recessive Mutation ◽  
Acid Mixture ◽  
Myocardial Cells ◽  
Lethal Mutant ◽  
Mexican Axolotl

A recessive mutation has been recently described in the Mexican Axolotl, Ambystoma mexicanum; in which the heart forms structurally, but does not contract (Humphrey, 1968. Anat. Rec. 160:475). In this study, the fine structure of myocardial cells from normal (+/+; +/c) and cardiac lethal mutant (c/c) embryos at Harrison's stage 40 was compared. The hearts were fixed in a 0.1 M phosphate buffered formaldehyde-glutaraldehyde-picric acid-styphnic acid mixture and were post fixed in 0.1 M s-collidine buffered 1% osmium tetroxide. A detailed study of heart development in normal and mutant embryos from stages 25-46 will be described elsewhere.

Cryogenic atomic force microscopy

1991 ◽  
Vol 49 ◽  
pp. 54-55
Author(s):  
K. A. Fisher ◽  
M. G. L. Gustafsson ◽  
M. B. Shattuck ◽  
J. Clarke
Keyword(s):  
Room Temperature ◽  
Rapid Freezing ◽  
Cryogenic Temperatures ◽  
Soft Or ◽  
Electrically Conductive ◽  
Force Microscopy ◽  
Flexible Molecules ◽  
Advantages And Disadvantages ◽  
Atomic Force ◽  
Chemical Perturbation

The atomic force microscope (AFM) is capable of imaging electrically conductive and non-conductive surfaces at atomic resolution. When used to image biological samples, however, lateral resolution is often limited to nanometer levels, due primarily to AFM tip/sample interactions. Several approaches to immobilize and stabilize soft or flexible molecules for AFM have been examined, notably, tethering coating, and freezing. Although each approach has its advantages and disadvantages, rapid freezing techniques have the special advantage of avoiding chemical perturbation, and minimizing physical disruption of the sample. Scanning with an AFM at cryogenic temperatures has the potential to image frozen biomolecules at high resolution. We have constructed a force microscope capable of operating immersed in liquid n-pentane and have tested its performance at room temperature with carbon and metal-coated samples, and at 143° K with uncoated ferritin and purple membrane (PM).

High-Resolution Scanning Electron Microscopy of Biological Specimens

1988 ◽  
Vol 46 ◽  
pp. 186-187
Author(s):  
M. Müller ◽  
R. Hermann
Keyword(s):  
High Resolution ◽  
Freeze Drying ◽  
Room Temperature ◽  
Structural Information ◽  
Freeze Substitution ◽  
Critical Point Drying ◽  
Sem Study ◽  
Major Factors ◽  
Structural Preservation ◽  
Preparation Techniques

Three major factors must be concomitantly assessed in order to extract relevant structural information from the surface of biological material at high resolution (2-3nm).Procedures based on chemical fixation and dehydration in graded solvent series seem inappropriate when aiming for TEM-like resolution. Cells inevitably shrink up to 30-70% of their initial volume during gehydration; important surface components e.g. glycoproteins may be lost. These problems may be circumvented by preparation techniques based on cryofixation. Freezedrying and freeze-substitution followed by critical point drying yields improved structural preservation in TEM. An appropriate preservation of dimensional integrity may be achieved by freeze-drying at - 85° C. The sample shrinks and may partially collapse as it is warmed to room temperature for subsequent SEM study. Observations at low temperatures are therefore a necessary prerequisite for high fidelity SEM. Compromises however have been unavoidable up until now. Aldehyde prefixation is frequently needed prior to freeze drying, rendering the sample resistant to treatment with distilled water.

Immunogold localization of HMB-45 antigen in pulmonary lymphangiomyomatosis

1995 ◽  
Vol 53 ◽  
pp. 998-999
Author(s):  
J.M. Minda ◽  
E. Dessy ◽  
G. G. Pietra
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Muscle Cells ◽  
Ultrastructural Localization ◽  
Reproductive Age ◽  
Thin Sections ◽  
Smooth Muscle Proliferation ◽  
Hmb 45

Pulmonary lymphangiomyomatosis (PLAM) is a rare disease occurring exclusively in women of reproductive age. It involves the lungs, lymph nodes and lymphatic ducts. In the lungs, it is characterized by the proliferation of smooth muscle cells around lymphatics in the bronchovascular bundles, lobular septa and pleura The nature of smooth muscle proliferation in PLAM is still unclear. Recently, reactivity of the smooth muscle cells for HMB-45, a melanoma-related antigen has been reported by immunohistochemistry. The purpose of this study was the ultrastructural localization of HMB-45 immunoreactivity in these cells using gold-labeled antibodies.Lung tissue from three cases of PLAM, referred to our Institution for lung transplantation, was embedded in either Poly/Bed 812 post-fixed in 1% osmium tetroxide, or in LR White, without osmication. For the immunogold technique, thin sections were placed on Nickel grids and incubated with affinity purified, monoclonal anti-melanoma antibody HMB-45 (1:1) (Enzo Diag. Co) overnight at 4°C. After extensive washing with PBS, grids were treated with Goat-anti-mouse-IgG-Gold (5nm) (1:10) (Amersham Life Sci) for 1 hour, at room temperature.

Opening images of synaptic vesicles and calcium localization by means of microwave fixation method

1990 ◽  
Vol 48 (2) ◽  
pp. 182-183
Author(s):  
Vinci Mizuhira ◽  
Hiroshi Hasegawa
Keyword(s):  
Synaptic Vesicles ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Sampling Procedure ◽  
Fixation Method ◽  
Potassium Oxalate ◽  
X Ray ◽  
Cacodylate Buffer ◽  
Ultrathin Sections ◽  
Microwave Fixation

Microwave irradiation (MWI) was applied to 0.3 to 1 cm3 blocks of rat central nervous system at 2.45 GHz/500W for about 20 sec in a fixative, at room temperature. Fixative composed of 2% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4, also contained 2 mM of CaCl2 , 1 mM of MgCl2, and 0.1% of tannic acid for conventional observation; and fuether 30-90 mM of potassium oxalate containing fixative was applied for the detection of calcium ion localization in cells. Tissue blocks were left in the same fixative for 30 to 180 min after MWI at room temperature, then proceeded to the sampling procedure, after postfixed with osmium tetroxide, embedded in Epon. Ultrathin sections were double stained with an useal manner. Oxalate treated sections were devided in two, stained and unstained one. The later oxalate treated unstained sections were analyzed with electron probe X-ray microanalyzer, the EDAX-PU-9800, at 40 KV accelerating voltage for 100 to 200 sec with point or selected area analyzing methods.

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