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.

Three Dimensional Immuno-Localization of Green Fluorescent Protein Chimeras Using Rapid Freezing and Freeze-Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 298-299
Author(s):  
Mary Morphew ◽  
David Mastronarde ◽  
Eileen O'Toole ◽  
Mark Ladinsky ◽  
Brad Marsh ◽  
...  
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Green Fluorescent ◽  
Structural Preservation

All microscopy is limited by the quality of the specimen under study. Three-dimensional (3-D) visualization of antigen localization using the electron microscope (EM) is particularly challenging due to the need to maintain the activity of some epitopes while preserving cellular ultrastructure. We have used rapid freezing to immobilize all cellular constituents almost instantaneously. Freeze-substitution of the frozen samples was used to stabilize the specimen and to accomplish low-temperature dehydration, minimizing perturbation of cellular structure. We have found that high pressure freezing, double jet freezing and plunge freezing are all useful for achieving high quality structural preservation for some cell types or for particular applications. For immunolocalization, we have had most success freeze-substituting into acetone containing 0.2% glutaraldehyde and 0.1 % uranyl acetate. We have utilized low-temperature acrylic embedding resins, Lowicryl HM20 and LRGold, to further maintain structure and decrease protein insolubility. Both of these resins have proven suitable for cutting serial thin sections.

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).

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°.

Membranous alterations in the cytoplasm and nucleus in a primitive neuroectodermal tumor of the brain

1978 ◽  
Vol 36 (2) ◽  
pp. 628-629
Author(s):  
C. N. Sun ◽  
C. Araoz ◽  
H. J. White
Keyword(s):  
Nuclear Envelope ◽  
Tumor Cells ◽  
Osmium Tetroxide ◽  
Primitive Neuroectodermal Tumor ◽  
Uranyl Acetate ◽  
Neuroectodermal Tumor ◽  
Annulate Lamellae ◽  
Lead Citrate ◽  
Thin Sections ◽  
The Brain

The ultrastructure of a cerebral primitive neuroectodermal tumor has been reported previously. In the present case, we will present some unusual previously unreported membranous structures and alterations in the cytoplasm and nucleus of the tumor cells.Specimens were cut into small pieces about 1 mm3 and immediately fixed in 4% glutaraldehyde in phosphate buffer for two hours, then post-fixed in 1% buffered osmium tetroxide for one hour. After dehydration, tissues were embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate.In the cytoplasm of the tumor cells, we found paired cisternae (Fig. 1) and annulate lamellae (Fig. 2) noting that the annulate lamellae were sometimes associated with the outer nuclear envelope (Fig. 3). These membranous structures have been reported in other tumor cells. In our case, mitochondrial to nuclear envelope fusions were often noted (Fig. 4). Although this phenomenon was reported in an oncocytoma, their frequency in the present study is quite striking.

Ultrastructure of myoepithelial cell in parotid gland

1988 ◽  
Vol 46 ◽  
pp. 342-343
Author(s):  
C. N. Sun
Keyword(s):  
Parotid Gland ◽  
Osmium Tetroxide ◽  
Individual Cell ◽  
Branching Processes ◽  
Muscle Cells ◽  
Myoepithelial Cells ◽  
Uranyl Acetate ◽  
Thin Sections ◽  
Gland Carcinoma ◽  
Parotid Gland Carcinoma

Myoepithelial cells have been observed in the prostate, harderian, apocrine, exocrine sweat and mammary glands. Such cells and their numerous branching processes form basket-like structures around the glandular acini. Their shapes are quite different from structures seen either in spindleshaped smooth muscle cells or skeletal muscle cells. These myoepithelial cells lie on the epithelial side of the basement membrane in the glands. This presentation describes the ultrastructure of such myoepithelial cells which have been found also in the parotid gland carcinoma from a 45-year old patient.Specimens were cut into small pieces about 1 mm3 and immediately fixed in 4 percent glutaraldehyde in phosphate buffer for two hours, then post-fixed in 1 percent buffered osmium tetroxide for 1 hour. After dehydration, tissues were embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate. Ultrastructurally, the pattern of each individual cell showed wide variations.

Observations of thick and thin sections in a field-emission SEM

1994 ◽  
Vol 52 ◽  
pp. 1018-1019
Author(s):  
W. P. Wergin ◽  
S. Roy ◽  
E. F. Erbe ◽  
C. A. Murphy ◽  
C. D. Pooley
Keyword(s):  
Electron Microscope ◽  
Field Emission ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Thin Sections ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope ◽  
Scanning Electron

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freezesubstituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures. Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's medium and sectioned for observation with a Hitachi S-4100 field emission scanning electron microscope that was equipped with an Oxford CT 1500 Cryotrans System. Thin sections, about 80 nm thick, similar to those generally used in conventional transmission electron microscope (TEM) studies were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min. Sections about 2 μm thick were also mounted and stained in a similar fashion. The grids were mounted on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at ambient temperature. Thick and thin sections of the larvae were evaluated and photographed in the SEM at different accelerating voltages. Figs. 4 and 5 have undergone contrast conversion so that the images would resemble transmitted electron micrographs obtained with a TEM.

Freeze substitution fixation of fungal reproductive structures and spores

1989 ◽  
Vol 47 ◽  
pp. 990-991
Author(s):  
C. W. Mims ◽  
E. A. Richardson
Keyword(s):  
Plant Pathogens ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Dialysis Membrane ◽  
Razor Blade ◽  
Thin Sections ◽  
Reproductive Structures ◽  
Dry Down ◽  
Diamond Knife

The advantages of freeze substitution fixation over conventional chemical fixation for preservation of ultrastructural details in fungi have been discussed by various authors. As most ascomycetes, basidiomycetes and deuteromycetes do not fix well using conventional chemical fixation protocols, freeze substitution has attracted the attention of many individuals interested in fungal ultrastructure. Thus far most workers using this technique on fungi have concentrated on thin walled somatic hyphae. However, in our laboratory we have experimented with the use of freeze substitution on a variety of fungal reproductive structures and spores with promising results.Here we present data on freeze substituted samples of sporangia of the zygomycete Umbellopsis vinacea, basidia of Exobasidium camelliae var. gracilis, developing teliospores of the smut Sporisorium sorghi, germinating teliospores of the rust Gymnosporangium clavipes, germinating conidia of the deuteromycete Cercosporidium personatum, and developing ascospores of Ascodesmis nigricans.Spores of G. clavipes and C. personatum were deposited on moist pieces of sterile dialysis membrane where they hydrated and germinated. Asci of A. nigricans developed on pieces of dialysis membrane lying on nutrient agar plates. U. vinacea was cultured on small pieces of agar-coated wire. In the plant pathogens E. camelliae var. gracilis and S. sorghi, a razor blade was used to remove smal1 pieces of infected host issue. All samples were plunged directly into liquid propane and processed for study according to Hoch.l Samples on dialysis membrane were flat embedded. Serial thin sections were cut using a diamond knife, collected on slot grids, and allowed to dry down onto Formvar coated aluminum racks. Sections were post stained with uranyl acetate and lead citrate.

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.

scholarly journals Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution

2022 ◽  
Author(s):  
Martin Schauflinger ◽  
Tim Bergner ◽  
Gregor Neusser ◽  
Christine Kranz ◽  
Clarissa Read
Keyword(s):  
High Pressure ◽  
Potassium Permanganate ◽  
Lipid Membranes ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Block Face ◽  
Critical Aspects

AbstractHigh-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

scholarly journals ULTRASTRUCTURAL INTERRELATIONSHIPS OF NERVE PROCESSES AND SMOOTH MUSCLE CELLS IN THREE DIMENSIONS

1966 ◽  
Vol 28 (1) ◽  
pp. 37-49 ◽  
Author(s):  
J. C. Thaemert
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Osmium Tetroxide ◽  
Three Dimensional ◽  
Muscle Cells ◽  
Uranyl Acetate ◽  
Intestinal Wall ◽  
Three Dimensions ◽  
Serial Sections ◽  
Muscularis Externa

The muscularis externa of the intestinal wall of frogs was fixed in osmium tetroxide, embedded in Vestopal-W, serially sectioned for electron microscopy, and stained with uranyl acetate. A method to obtain individually mounted and properly positioned serial sections is described. The three-dimensional techniques used during the course of this investigation demonstrate that it is possible to examine carefully relatively large areas of tissue on individual serial sections with the electron microscope and subsequently to construct montages of electron micrographs of pertinent areas from each section. Several carefully rendered interrelationships of nerve processes and smooth muscle cells in three dimensions are exhibited and described. Recent studies of other neuro-effector relationships are discussed in relation to the present status of the nature and organization of the autonomic nervous system in visceral organs.

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